Axmi-115, axmi-113, axmi-005, axmi-163 and axmi-184: insecticidal proteins and methods for their use

ABSTRACT

Compositions and methods for conferring insecticidal activity to host cells are provided. Compositions comprising a coding sequence for a delta-endotoxin polypeptide are provided. The coding sequences can be used in DNA constructs or expression cassettes for transformation and expression in host cells. Compositions also comprise transformed host cells. In particular, isolated delta-endotoxin nucleic acid molecules are provided. Additionally, amino acid sequences corresponding to the polynucleotides are encompassed, and antibodies specifically binding to those amino acid sequences. In particular, the present invention provides for isolated nucleic acid molecules comprising nucleotide sequences encoding the amino acid sequence shown in SEQ ID NO:4, 5, 6, 13, or 14, or the nucleotide sequence set forth in SEQ ID NO:1, 2, 3, 11, or 12, as well as variants and fragments thereof.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.12/497,221, filed Jul. 2, 2009, which claims the benefit of U.S.Provisional Application Ser. No. 61/077,812, filed Jul. 2, 2008, andU.S. Provisional Application Ser. No. 61/158,953, filed Mar. 10, 2009,the contents of which are hereby incorporated in their entirety byreference herein.

REFERENCE TO SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB

The official copy of the sequence listing is submitted concurrently withthe specification as an ASCII formatted text file via EFS-Web, with afile name of “APA058US01NSEQLIST.txt”, a creation date of Nov. 9, 2012,and a size of 88 kilobytes. The sequence listing filed via EFS-Web ispart of the specification and is hereby incorporated in its entirety byreference herein.

FIELD OF THE INVENTION

This invention relates to the field of molecular biology. Provided arenovel genes that encode insecticidal proteins. These proteins and thenucleic acid sequences that encode them are useful in preparinginsecticidal formulations and in the production of transgenicinsect-resistant plants.

BACKGROUND OF THE INVENTION

Bacillus thuringiensis is a Gram-positive spore forming soil bacteriumcharacterized by its ability to produce crystalline inclusions that arespecifically toxic to certain orders and species of insects, but areharmless to plants and other non-targeted organisms. For this reason,compositions including Bacillus thuringiensis strains or theirinsecticidal proteins can be used as environmentally-acceptableinsecticides to control agricultural insect pests or insect vectors fora variety of human or animal diseases.

Crystal (Cry) proteins (delta-endotoxins) from Bacillus thuringiensishave potent insecticidal activity against predominantly Lepidopteran,Dipteran, and Coleopteran larvae. These proteins also have shownactivity against Hymenoptera, Homoptera, Phthiraptera, Mallophaga, andAcari pest orders, as well as other invertebrate orders such asNemathelminthes, Platyhelminthes, and Sarcomastigorphora (Feitelson(1993) The Bacillus Thuringiensis family tree. In Advanced EngineeredPesticides, Marcel Dekker, Inc., New York, N.Y.) These proteins wereoriginally classified as CryI to CryV based primarily on theirinsecticidal activity. The major classes were Lepidoptera-specific (I),Lepidoptera- and Diptera-specific (II), Coleoptera-specific (III),Diptera-specific (IV), and nematode-specific (V) and (VI). The proteinswere further classified into subfamilies; more highly related proteinswithin each family were assigned divisional letters such as Cry1A,Cry1B, Cry1C, etc. Even more closely related proteins within eachdivision were given names such as Cry1C1, C1C2, etc.

A new nomenclature was recently described for the Cry genes based uponamino acid sequence homology rather than insect target specificity(Crickmore et al. (1998) Microbiol. Mol. Biol. Rev. 62:807-813). In thenew classification, each toxin is assigned a unique name incorporating aprimary rank (an Arabic number), a secondary rank (an uppercase letter),a tertiary rank (a lowercase letter), and a quaternary rank (anotherArabic number). In the new classification, Roman numerals have beenexchanged for Arabic numerals in the primary rank. Proteins with lessthan 45% sequence identity have different primary ranks, and thecriteria for secondary and tertiary ranks are 78% and 95%, respectively.

The crystal protein does not exhibit insecticidal activity until it hasbeen ingested and solubilized in the insect midgut. The ingestedprotoxin is hydrolyzed by proteases in the insect digestive tract to anactive toxic molecule. (Höfte and Whiteley (1989) Microbiol. Rev.53:242-255). This toxin binds to apical brush border receptors in themidgut of the target larvae and inserts into the apical membranecreating ion channels or pores, resulting in larval death.

Delta-endotoxins generally have five conserved sequence domains, andthree conserved structural domains (see, for example, de Maagd et al.(2001) Trends Genetics 17:193-199). The first conserved structuraldomain consists of seven alpha helices and is involved in membraneinsertion and pore formation. Domain II consists of three beta-sheetsarranged in a “Greek key” configuration, and domain III consists of twoantiparallel beta-sheets in “jelly-roll” formation (de Maagd et al.,2001, supra). Domains II and III are involved in receptor recognitionand binding, and are therefore considered determinants of toxinspecificity.

Aside from delta-endotoxins, there are several other known classes ofpesticidal protein toxins. The VIP1/VIP2 toxins (see, for example, U.S.Pat. No. 5,770,696) are binary pesticidal toxins that exhibit strongactivity on insects by a mechanism believed to involve receptor-mediatedendocytosis followed by cellular toxification, similar to the mode ofaction of other binary (“A/B”) toxins. A/B toxins such as VIP, C2, CDT,CST, or the B. anthracis edema and lethal toxins initially interact withtarget cells via a specific, receptor-mediated binding of “B” componentsas monomers. These monomers then form homoheptamers. The “B”heptamer-receptor complex then acts as a docking platform thatsubsequently binds and allows the translocation of an enzymatic “A”component(s) into the cytosol via receptor-mediated endocytosis. Onceinside the cell's cytosol, “A” components inhibit normal cell functionby, for example, ADP-ribosylation of G-actin, or increasingintracellular levels of cyclic AMP (cAMP). See Barth et al. (2004)Microbiol Mol Biol Rev 68:373-402.

The intensive use of B. thuringiensis-based insecticides has alreadygiven rise to resistance in field populations of the diamondback moth,Plutella xylostella (Ferré and Van Rie (2002) Annu. Rev. Entomol.47:501-533). The most common mechanism of resistance is the reduction ofbinding of the toxin to its specific midgut receptor(s). This may alsoconfer cross-resistance to other toxins that share the same receptor(Ferré and Van Rie (2002)).

SUMMARY OF INVENTION

Compositions and methods for conferring insect resistance to bacteria,plants, plant cells, tissues and seeds are provided. Compositionsinclude nucleic acid molecules encoding sequences for delta-endotoxinpolypeptides, vectors comprising those nucleic acid molecules, and hostcells comprising the vectors. Compositions also include the polypeptidesequences of the endotoxin, and antibodies to those polypeptides. Thenucleotide sequences can be used in DNA constructs or expressioncassettes for transformation and expression in organisms, includingmicroorganisms and plants. The nucleotide or amino acid sequences may besynthetic sequences that have been designed for expression in anorganism including, but not limited to, a microorganism or a plant.Compositions also comprise transformed bacteria, plants, plant cells,tissues, and seeds.

In particular, isolated nucleic acid molecules corresponding todelta-endotoxin nucleic acid sequences are provided. Additionally, aminoacid sequences corresponding to the polynucleotides are encompassed. Inparticular, the present invention provides for an isolated nucleic acidmolecule comprising a nucleotide sequence encoding the amino acidsequence shown in any of SEQ ID NO:4, 5, 6, 13, or 14, or a nucleotidesequence set forth in any of SEQ ID NO:1, 2, 3, 11, or 12, as well asvariants and fragments thereof. Nucleotide sequences that arecomplementary to a nucleotide sequence of the invention, or thathybridize to a sequence of the invention are also encompassed.

The compositions and methods of the invention are useful for theproduction of organisms with insecticide resistance, specificallybacteria and plants. These organisms and compositions derived from themare desirable for agricultural purposes. The compositions of theinvention are also useful for generating altered or improveddelta-endotoxin proteins that have insecticidal activity, or fordetecting the presence of delta-endotoxin proteins or nucleic acids inproducts or organisms.

The following embodiments are encompassed by the present invention:

1. An isolated nucleic acid molecule comprising a nucleotide sequenceselected from the group consisting of:

-   -   a) the nucleotide sequence of any of SEQ ID NO:1, 2, 3, 11, or        12, or a complement thereof;    -   b) a nucleotide sequence that encodes a polypeptide comprising        the amino acid sequence of any of SEQ ID NO:4, 5, 6, 13, or 14;    -   c) a nucleotide sequence that encodes a polypeptide comprising        an amino acid sequence having at least 96% sequence identity to        the amino acid sequence of SEQ ID NO:4 or 14, wherein said amino        acid sequence has insecticidal activity;    -   d) a nucleotide sequence that encodes a polypeptide comprising        an amino acid sequence having at least 95% sequence identity to        the amino acid sequence of SEQ ID NO:6, wherein said amino acid        sequence has insecticidal activity; and    -   e) a nucleotide sequence encoding an insecticidal polypeptide        that is a variant of SEQ ID NO:4, 5, 6, 13, or 14, wherein said        variant is the result of one or more domain(s) of SEQ ID NO:4,        5, 6, 13, or 14 being exchanged with the corresponding domain(s)        of SEQ ID NO:4, 5, 6, 13, or 14, wherein said nucleotide        sequence encodes an insecticidal polypeptide.

2. The isolated nucleic acid molecule of embodiment 1, wherein saidnucleotide sequence is a synthetic sequence that has been designed forexpression in a plant.

3. The isolated nucleic acid molecule of embodiment 2, wherein saidnucleotide sequence is selected from the group consisting of SEQ IDNO:15, 16, 17, and 18.

4. An expression cassette comprising the nucleic acid molecule ofembodiment 1.

5. The expression cassette of embodiment 4, further comprising a nucleicacid molecule encoding a heterologous polypeptide.

6. A host cell that contains the expression cassette of embodiment 4.

7. The host cell of embodiment 6 that is a bacterial host cell.

8. The host cell of embodiment 6 that is a plant cell.

9. A transgenic plant comprising the host cell of embodiment 8.

10. The transgenic plant of embodiment 9, wherein said plant is selectedfrom the group consisting of maize, sorghum, wheat, cabbage, sunflower,tomato, crucifers, peppers, potato, cotton, rice, soybean, sugarbeet,sugarcane, tobacco, barley, and oilseed rape.

11. An isolated polypeptide with insecticidal activity, selected fromthe group consisting of:

-   -   a) a polypeptide comprising the amino acid sequence of any of        SEQ ID NO:4, 5, 6, 13, or 14;    -   b) a polypeptide comprising an amino acid sequence having at        least 96% sequence identity to the amino acid sequence of SEQ ID        NO:4 or 14, wherein said amino acid sequence has insecticidal        activity;    -   c) a polypeptide comprising an amino acid sequence having at        least 95% sequence identity to the amino acid sequence of SEQ ID        NO:6, wherein said amino acid sequence has insecticidal        activity;    -   d) a polypeptide that is encoded by the nucleotide sequence of        any of SEQ ID NO:1, 2, 3, 11, or 12; and    -   e) a polypeptide that is a variant of SEQ ID NO:4, 5, 6, 13, or        14, wherein said variant is the result of one or more domain(s)        of SEQ ID NO:4, 5, 6, 13, or 14 being exchanged with the        corresponding domain(s) of SEQ ID NO:4, 5, 6, 13, or 14, wherein        said polypeptide has insecticidal activity.

12. The polypeptide of embodiment 11 further comprising heterologousamino acid sequences.

13. An antibody that selectively binds to the polypeptide of embodiment11.

14. A composition comprising the polypeptide of embodiment 11.

15. The composition of embodiment 14, wherein said composition isselected from the group consisting of a powder, dust, pellet, granule,spray, emulsion, colloid, and solution.

16. The composition of embodiment 14, wherein said composition isprepared by desiccation, lyophilization, homogenization, extraction,filtration, centrifugation, sedimentation, or concentration of a cultureof Bacillus thuringiensis cells.

17. The composition of embodiment 14, comprising from about 1% to about99% by weight of said polypeptide.

18. A method for controlling a lepidopteran or coleopteran pestpopulation comprising contacting said population with aninsecticidally-effective amount of the polypeptide of embodiment 11.

19. A method for killing a lepidopteran or coleopteran pest, comprisingcontacting said pest with, or feeding to said pest, aninsecticidally-effective amount of the polypeptide of embodiment 11.

20. A method for producing a polypeptide with insecticidal activity,comprising culturing the host cell of embodiment 6 under conditions inwhich the nucleic acid molecule encoding the polypeptide is expressed.

21. A plant having stably incorporated into its genome a DNA constructcomprising a nucleotide sequence that encodes a protein havinginsecticidal activity, wherein said nucleotide sequence is selected fromthe group consisting of:

-   -   a) the nucleotide sequence of any of SEQ ID NO:1, 2, 3, 11, or        12;    -   b) a nucleotide sequence that encodes a polypeptide comprising        the amino acid sequence of any of SEQ ID NO:4, 5, 6, 13, or 14;    -   c) a nucleotide sequence that encodes a polypeptide comprising        an amino acid sequence having at least 96% sequence identity to        the amino acid sequence of SEQ ID NO:4 or 14, wherein said amino        acid sequence has insecticidal activity;    -   d) a nucleotide sequence that encodes a polypeptide comprising        an amino acid sequence having at least 95% sequence identity to        the amino acid sequence of SEQ ID NO:6, wherein said amino acid        sequence has insecticidal activity; and    -   e) a nucleotide sequence encoding an insecticidal polypeptide        that is a variant of SEQ ID NO:4, 5, 6, 13, or 14, wherein said        variant is the result of one or more domain(s) of SEQ ID NO:4,        5, 6, 13, or 14 being exchanged with the corresponding domain(s)        of SEQ ID NO:4, 5, 6, 13, or 14, wherein said nucleotide        sequence encodes an insecticidal polypeptide;        wherein said nucleotide sequence is operably linked to a        promoter that drives expression of a coding sequence in a plant        cell.

22. The plant of embodiment 21, wherein said plant is a plant cell.

23. A transgenic seed of the plant of embodiment 21, wherein said seedcomprises a nucleotide sequence selected from the group consisting of:

-   -   a) the nucleotide sequence of any of SEQ ID NO:1, 2, 3, 11, or        12;    -   b) a nucleotide sequence that encodes a polypeptide comprising        the amino acid sequence of any of SEQ ID NO:4, 5, 6, 13, or 14;    -   c) a nucleotide sequence that encodes a polypeptide comprising        an amino acid sequence having at least 96% sequence identity to        the amino acid sequence of SEQ ID NO:4 or 14, wherein said amino        acid sequence has insecticidal activity;    -   d) a nucleotide sequence that encodes a polypeptide comprising        an amino acid sequence having at least 95% sequence identity to        the amino acid sequence of SEQ ID NO:6, wherein said amino acid        sequence has insecticidal activity; and    -   e) a nucleotide sequence encoding an insecticidal polypeptide        that is a variant of SEQ ID NO:4, 5, 6, 13, or 14, wherein said        variant is the result of one or more domain(s) of SEQ ID NO:4,        5, 6, 13, or 14 being exchanged with the corresponding domain(s)        of SEQ ID NO:4, 5, 6, 13, or 14, wherein said nucleotide        sequence encodes an insecticidal polypeptide.

24. A method for protecting a plant from an insect pest, comprisingintroducing into said plant or cell thereof at least one expressionvector comprising a nucleotide sequence that encodes an insecticidalpolypeptide, wherein said nucleotide sequence is selected from the groupconsisting of:

-   -   a) the nucleotide sequence of any of SEQ ID NO:1, 2, 3, 11, or        12;    -   b) a nucleotide sequence that encodes a polypeptide comprising        the amino acid sequence of any of SEQ ID NO:4, 5, 6, 13, or 14;    -   c) a nucleotide sequence that encodes a polypeptide comprising        an amino acid sequence having at least 96% sequence identity to        the amino acid sequence of SEQ ID NO:4 or 14, wherein said amino        acid sequence has insecticidal activity;    -   d) a nucleotide sequence that encodes a polypeptide comprising        an amino acid sequence having at least 95% sequence identity to        the amino acid sequence of SEQ ID NO:6, wherein said amino acid        sequence has insecticidal activity; and    -   e) a nucleotide sequence encoding an insecticidal polypeptide        that is a variant of SEQ ID NO:4, 5, 6, 13, or 14, wherein said        variant is the result of one or more domain(s) of SEQ ID NO:4,        5, 6, 13, or 14 being exchanged with the corresponding domain(s)        of SEQ ID NO:4, 5, 6, 13, or 14, wherein said nucleotide        sequence encodes an insecticidal polypeptide.

25. The method of embodiment 24, wherein said plant produces aninsecticidal polypeptide having insecticidal activity against alepidopteran or coleopteran pest.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B show an alignment of AXMI-113 (SEQ ID NO:5), AXMI-005(SEQ ID NO:4), and AXMI-115 (SEQ ID NO:6). The left and right arrowsmark the boundaries of the C-terminal ⅓^(rd) region of the proteins.

FIG. 2 depicts domains within AXMI-005 and AXMI-115 that are swapped togenerate new toxins.

DETAILED DESCRIPTION

The present invention is drawn to compositions and methods forregulating insect resistance in organisms, particularly plants or plantcells. The methods involve transforming organisms with a nucleotidesequence encoding a delta-endotoxin protein of the invention. Inparticular, the nucleotide sequences of the invention are useful forpreparing plants and microorganisms that possess insecticidal activity.Thus, transformed bacteria, plants, plant cells, plant tissues and seedsare provided. Compositions are delta-endotoxin nucleic acids andproteins of Bacillus thuringiensis. The sequences find use in theconstruction of expression vectors for subsequent transformation intoorganisms of interest, as probes for the isolation of otherdelta-endotoxin genes, and for the generation of altered insecticidalproteins by methods known in the art, such as domain swapping or DNAshuffling. See, for example, Table 2 and FIG. 2. The proteins find usein controlling or killing lepidopteran, coleopteran, and other insectpopulations, and for producing compositions with insecticidal activity.

By “delta-endotoxin” is intended a toxin from Bacillus thuringiensisthat has toxic activity against one or more pests, including, but notlimited to, members of the Lepidoptera, Diptera, and Coleoptera orders,or a protein that has homology to such a protein. In some cases,delta-endotoxin proteins have been isolated from other organisms,including Clostridium bifermentans and Paenibacillus popilliae.Delta-endotoxin proteins include amino acid sequences deduced from thefull-length nucleotide sequences disclosed herein, and amino acidsequences that are shorter than the full-length sequences, either due tothe use of an alternate downstream start site, or due to processing thatproduces a shorter protein having insecticidal activity. Processing mayoccur in the organism the protein is expressed in, or in the pest afteringestion of the protein.

Delta-endotoxins include proteins identified as cry1 through cry43, cyt1and cyt2, and Cyt-like toxin. There are currently over 250 known speciesof delta-endotoxins with a wide range of specificities and toxicities.For an expansive list see Crickmore et al. (1998), Microbiol. Mol. Biol.Rev. 62:807-813, and for regular updates see Crickmore et al. (2003)“Bacillus thuringiensis toxin nomenclature,” atwww.biols.susx.ac.uk/Home/Neil_Crickmore/Bt/index.

Provided herein are novel isolated nucleotide sequences that conferinsecticidal activity. Also provided are the amino acid sequences of thedelta-endotoxin proteins. The protein resulting from translation of thisgene allows cells to control or kill insects that ingest it.

Isolated Nucleic Acid Molecules, and Variants and Fragments Thereof

One aspect of the invention pertains to isolated or recombinant nucleicacid molecules comprising nucleotide sequences encoding delta-endotoxinproteins and polypeptides or biologically active portions thereof, aswell as nucleic acid molecules sufficient for use as hybridizationprobes to identify delta-endotoxin encoding nucleic acids. As usedherein, the term “nucleic acid molecule” is intended to include DNAmolecules (e.g., recombinant DNA, cDNA or genomic DNA) and RNA molecules(e.g., mRNA) and analogs of the DNA or RNA generated using nucleotideanalogs. The nucleic acid molecule can be single-stranded ordouble-stranded, but preferably is double-stranded DNA.

An “isolated” or “purified” nucleic acid molecule or protein, orbiologically active portion thereof, is substantially free of othercellular material, or culture medium when produced by recombinanttechniques, or substantially free of chemical precursors or otherchemicals when chemically synthesized. Preferably, an “isolated” nucleicacid is free of sequences (preferably protein encoding sequences) thatnaturally flank the nucleic acid (i.e., sequences located at the 5′ and3′ ends of the nucleic acid) in the genomic DNA of the organism fromwhich the nucleic acid is derived. For purposes of the invention,“isolated” when used to refer to nucleic acid molecules excludesisolated chromosomes. For example, in various embodiments, the isolateddelta-endotoxin encoding nucleic acid molecule can contain less thanabout 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotidesequences that naturally flank the nucleic acid molecule in genomic DNAof the cell from which the nucleic acid is derived. A delta-endotoxinprotein that is substantially free of cellular material includespreparations of protein having less than about 30%, 20%, 10%, or 5% (bydry weight) of non-delta-endotoxin protein (also referred to herein as a“contaminating protein”).

Nucleotide sequences encoding the proteins of the present inventioninclude the sequence set forth in SEQ ID NO:1, 2, 3, 11, or 12, andvariants, fragments, and complements thereof. By “complement” isintended a nucleotide sequence that is sufficiently complementary to agiven nucleotide sequence such that it can hybridize to the givennucleotide sequence to thereby form a stable duplex. The correspondingamino acid sequence for the delta-endotoxin protein encoded by thisnucleotide sequence are set forth in SEQ ID NO:4, 5, 6, 13, or 14.

Nucleic acid molecules that are fragments of these delta-endotoxinencoding nucleotide sequences are also encompassed by the presentinvention. By “fragment” is intended a portion of the nucleotidesequence encoding a delta-endotoxin protein. A fragment of a nucleotidesequence may encode a biologically active portion of a delta-endotoxinprotein, or it may be a fragment that can be used as a hybridizationprobe or PCR primer using methods disclosed below. Nucleic acidmolecules that are fragments of a delta-endotoxin nucleotide sequencecomprise at least about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900,1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550,1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2150,2200, 2250, 2300, 2350, 2400, 2450, 2500, 2550, 2600, 2650, 2700, 2750,2800, 2850, 2900, 2950, 3000, 3050, 3100, 3150, 3200, 3250, 3300, 3350contiguous nucleotides, or up to the number of nucleotides present in afull-length delta-endotoxin encoding nucleotide sequence disclosedherein depending upon the intended use. By “contiguous” nucleotides isintended nucleotide residues that are immediately adjacent to oneanother. Fragments of the nucleotide sequences of the present inventionwill encode protein fragments that retain the biological activity of thedelta-endotoxin protein and, hence, retain insecticidal activity. By“retains activity” is intended that the fragment will have at leastabout 30%, at least about 50%, at least about 70%, 80%, 90%, 95% orhigher of the insecticidal activity of the delta-endotoxin protein.Methods for measuring insecticidal activity are well known in the art.See, for example, Czapla and Lang (1990) J. Econ. Entomol. 83:2480-2485;Andrews et al. (1988) Biochem. J. 252:199-206; Marrone et al. (1985) J.of Economic Entomology 78:290-293; and U.S. Pat. No. 5,743,477, all ofwhich are herein incorporated by reference in their entirety.

A fragment of a delta-endotoxin encoding nucleotide sequence thatencodes a biologically active portion of a protein of the invention willencode at least about 15, 25, 30, 50, 75, 100, 125, 150, 175, 200, 250,300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950,1000, 1050, 1100 contiguous amino acids, or up to the total number ofamino acids present in a full-length delta-endotoxin protein of theinvention.

Preferred delta-endotoxin proteins of the present invention are encodedby a nucleotide sequence sufficiently identical to the nucleotidesequence of SEQ ID NO:1, 2, 3, 11, or 12. By “sufficiently identical” isintended an amino acid or nucleotide sequence that has at least about60% or 65% sequence identity, about 70% or 75% sequence identity, about80% or 85% sequence identity, about 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or greater sequence identity compared to a referencesequence using one of the alignment programs described herein usingstandard parameters. One of skill in the art will recognize that thesevalues can be appropriately adjusted to determine corresponding identityof proteins encoded by two nucleotide sequences by taking into accountcodon degeneracy, amino acid similarity, reading frame positioning, andthe like.

To determine the percent identity of two amino acid sequences or of twonucleic acids, the sequences are aligned for optimal comparisonpurposes. The percent identity between the two sequences is a functionof the number of identical positions shared by the sequences (i.e.,percent identity=number of identical positions/total number of positions(e.g., overlapping positions)×100). In one embodiment, the two sequencesare the same length. In another embodiment, the comparison is across theentirety of the reference sequence (e.g., across the entirety of one ofSEQ ID NO:1, 2, 3, 11, or 12, or across the entirety of one of SEQ IDNO:4, 5, 6, 13, or 14). The percent identity between two sequences canbe determined using techniques similar to those described below, with orwithout allowing gaps. In calculating percent identity, typically exactmatches are counted.

The determination of percent identity between two sequences can beaccomplished using a mathematical algorithm. A nonlimiting example of amathematical algorithm utilized for the comparison of two sequences isthe algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA87:2264, modified as in Karlin and Altschul (1993) Proc. Natl. Acad.Sci. USA 90:5873-5877. Such an algorithm is incorporated into the BLASTNand BLASTX programs of Altschul et al. (1990) J. Mol. Biol. 215:403.BLAST nucleotide searches can be performed with the BLASTN program,score=100, wordlength=12, to obtain nucleotide sequences homologous todelta-endotoxin-like nucleic acid molecules of the invention. BLASTprotein searches can be performed with the BLASTX program, score=50,wordlength=3, to obtain amino acid sequences homologous todelta-endotoxin protein molecules of the invention. To obtain gappedalignments for comparison purposes, Gapped BLAST (in BLAST 2.0) can beutilized as described in Altschul et al. (1997) Nucleic Acids Res.25:3389. Alternatively, PSI-Blast can be used to perform an iteratedsearch that detects distant relationships between molecules. SeeAltschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST, andPSI-Blast programs, the default parameters of the respective programs(e.g., BLASTX and BLASTN) can be used. Alignment may also be performedmanually by inspection.

Another non-limiting example of a mathematical algorithm utilized forthe comparison of sequences is the ClustalW algorithm (Higgins et al.(1994) Nucleic Acids Res. 22:4673-4680). ClustalW compares sequences andaligns the entirety of the amino acid or DNA sequence, and thus canprovide data about the sequence conservation of the entire amino acidsequence. The ClustalW algorithm is used in several commerciallyavailable DNA/amino acid analysis software packages, such as the ALIGNXmodule of the Vector NTI Program Suite (Invitrogen Corporation,Carlsbad, Calif.). After alignment of amino acid sequences withClustalW, the percent amino acid identity can be assessed. Anon-limiting example of a software program useful for analysis ofClustalW alignments is GENEDOC™. GENEDOC™ (Karl Nicholas) allowsassessment of amino acid (or DNA) similarity and identity betweenmultiple proteins. Another non-limiting example of a mathematicalalgorithm utilized for the comparison of sequences is the algorithm ofMyers and Miller (1988) CABIOS 4:11-17. Such an algorithm isincorporated into the ALIGN program (version 2.0), which is part of theGCG Wisconsin Genetics Software Package, Version 10 (available fromAccelrys, Inc., 9685 Scranton Rd., San Diego, Calif., USA). Whenutilizing the ALIGN program for comparing amino acid sequences, a PAM120weight residue table, a gap length penalty of 12, and a gap penalty of 4can be used.

Unless otherwise stated, GAP Version 10, which uses the algorithm ofNeedleman and Wunsch (1970) J. Mol. Biol. 48(3):443-453, will be used todetermine sequence identity or similarity using the followingparameters: % identity and % similarity for a nucleotide sequence usingGAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmp scoringmatrix; % identity or % similarity for an amino acid sequence using GAPweight of 8 and length weight of 2, and the BLOSUM62 scoring program.Equivalent programs may also be used. By “equivalent program” isintended any sequence comparison program that, for any two sequences inquestion, generates an alignment having identical nucleotide residuematches and an identical percent sequence identity when compared to thecorresponding alignment generated by GAP Version 10.

The invention also encompasses variant nucleic acid molecules.“Variants” of the delta-endotoxin encoding nucleotide sequences includethose sequences that encode the delta-endotoxin proteins disclosedherein but that differ conservatively because of the degeneracy of thegenetic code as well as those that are sufficiently identical asdiscussed above. Naturally occurring allelic variants can be identifiedwith the use of well-known molecular biology techniques, such aspolymerase chain reaction (PCR) and hybridization techniques as outlinedbelow. Variant nucleotide sequences also include synthetically derivednucleotide sequences that have been generated, for example, by usingsite-directed mutagenesis but which still encode the delta-endotoxinproteins disclosed in the present invention as discussed below. Variantproteins encompassed by the present invention are biologically active,that is they continue to possess the desired biological activity of thenative protein, that is, retaining insecticidal activity. By “retainsactivity” is intended that the variant will have at least about 30%, atleast about 50%, at least about 70%, or at least about 80% of theinsecticidal activity of the native protein. Methods for measuringinsecticidal activity are well known in the art. See, for example,Czapla and Lang (1990) J. Econ. Entomol. 83: 2480-2485; Andrews et al.(1988) Biochem. J. 252:199-206; Marrone et al. (1985) J. of EconomicEntomology 78:290-293; and U.S. Pat. No. 5,743,477, all of which areherein incorporated by reference in their entirety.

The skilled artisan will further appreciate that changes can beintroduced by mutation of the nucleotide sequences of the inventionthereby leading to changes in the amino acid sequence of the encodeddelta-endotoxin proteins, without altering the biological activity ofthe proteins. Thus, variant isolated nucleic acid molecules can becreated by introducing one or more nucleotide substitutions, additions,or deletions into the corresponding nucleotide sequence disclosedherein, such that one or more amino acid substitutions, additions ordeletions are introduced into the encoded protein. Mutations can beintroduced by standard techniques, such as site-directed mutagenesis andPCR-mediated mutagenesis. Such variant nucleotide sequences are alsoencompassed by the present invention.

For example, conservative amino acid substitutions may be made at one ormore predicted, nonessential amino acid residues. A “nonessential” aminoacid residue is a residue that can be altered from the wild-typesequence of a delta-endotoxin protein without altering the biologicalactivity, whereas an “essential” amino acid residue is required forbiological activity. A “conservative amino acid substitution” is one inwhich the amino acid residue is replaced with an amino acid residuehaving a similar side chain. Families of amino acid residues havingsimilar side chains have been defined in the art. These families includeamino acids with basic side chains (e.g., lysine, arginine, histidine),acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polarside chains (e.g., glycine, asparagine, glutamine, serine, threonine,tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine).

Delta-endotoxins generally have five conserved sequence domains, andthree conserved structural domains (see, for example, de Maagd et al.(2001) Trends Genetics 17:193-199). The first conserved structuraldomain consists of seven alpha helices and is involved in membraneinsertion and pore formation. Domain II consists of three beta-sheetsarranged in a Greek key configuration, and domain III consists of twoantiparallel beta-sheets in “jelly-roll” formation (de Maagd et al.,2001, supra). Domains II and III are involved in receptor recognitionand binding, and are therefore considered determinants of toxinspecificity.

Amino acid substitutions may be made in nonconserved regions that retainfunction. In general, such substitutions would not be made for conservedamino acid residues, or for amino acid residues residing within aconserved motif, where such residues are essential for protein activity.Examples of residues that are conserved and that may be essential forprotein activity include, for example, residues that are identicalbetween all proteins contained in an alignment of the amino acidsequences of the present invention and known delta-endotoxin sequences.Examples of residues that are conserved but that may allow conservativeamino acid substitutions and still retain activity include, for example,residues that have only conservative substitutions between all proteinscontained in an alignment of the amino acid sequences of the presentinvention and known delta-endotoxin sequences. However, one of skill inthe art would understand that functional variants may have minorconserved or nonconserved alterations in the conserved residues.

Alternatively, variant nucleotide sequences can be made by introducingmutations randomly along all or part of the coding sequence, such as bysaturation mutagenesis, and the resultant mutants can be screened forability to confer delta-endotoxin activity to identify mutants thatretain activity. Following mutagenesis, the encoded protein can beexpressed recombinantly, and the activity of the protein can bedetermined using standard assay techniques.

Using methods such as PCR, hybridization, and the like correspondingdelta-endotoxin sequences can be identified, such sequences havingsubstantial identity to the sequences of the invention. See, forexample, Sambrook and Russell (2001) Molecular Cloning: A LaboratoryManual. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.)and Innis, et al. (1990) PCR Protocols: A Guide to Methods andApplications (Academic Press, NY).

In a hybridization method, all or part of the delta-endotoxin nucleotidesequence can be used to screen cDNA or genomic libraries. Methods forconstruction of such cDNA and genomic libraries are generally known inthe art and are disclosed in Sambrook and Russell, 2001, supra. Theso-called hybridization probes may be genomic DNA fragments, cDNAfragments, RNA fragments, or other oligonucleotides, and may be labeledwith a detectable group such as ³²P, or any other detectable marker,such as other radioisotopes, a fluorescent compound, an enzyme, or anenzyme co-factor. Probes for hybridization can be made by labelingsynthetic oligonucleotides based on the known delta-endotoxin-encodingnucleotide sequence disclosed herein. Degenerate primers designed on thebasis of conserved nucleotides or amino acid residues in the nucleotidesequence or encoded amino acid sequence can additionally be used. Theprobe typically comprises a region of nucleotide sequence thathybridizes under stringent conditions to at least about 12, at leastabout 25, at least about 50, 75, 100, 125, 150, 175, 200, 250, 300, 350,or 400 consecutive nucleotides of delta-endotoxin encoding nucleotidesequence of the invention or a fragment or variant thereof. Methods forthe preparation of probes for hybridization are generally known in theart and are disclosed in Sambrook and Russell, 2001, supra hereinincorporated by reference.

For example, an entire delta-endotoxin sequence disclosed herein, or oneor more portions thereof, may be used as a probe capable of specificallyhybridizing to corresponding delta-endotoxin-like sequences andmessenger RNAs. To achieve specific hybridization under a variety ofconditions, such probes include sequences that are unique and arepreferably at least about 10 nucleotides in length, or at least about 20nucleotides in length. Such probes may be used to amplify correspondingdelta-endotoxin sequences from a chosen organism by PCR. This techniquemay be used to isolate additional coding sequences from a desiredorganism or as a diagnostic assay to determine the presence of codingsequences in an organism. Hybridization techniques include hybridizationscreening of plated DNA libraries (either plaques or colonies; see, forexample, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual(2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).

Hybridization of such sequences may be carried out under stringentconditions. By “stringent conditions” or “stringent hybridizationconditions” is intended conditions under which a probe will hybridize toits target sequence to a detectably greater degree than to othersequences (e.g., at least 2-fold over background). Stringent conditionsare sequence-dependent and will be different in depending uponcircumstances. By controlling the stringency of the hybridization and/orwashing conditions, target sequences that are 100% complementary to theprobe can be identified (homologous probing). Alternatively, stringencyconditions can be adjusted to allow some mismatching in sequences sothat lower degrees of similarity are detected (heterologous probing).Generally, a probe is less than about 1000 nucleotides in length,preferably less than 500 nucleotides in length.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., anda wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a wash in 0.1×SSC at 60 to 65° C. Optionally, wash buffersmay comprise about 0.1% to about 1% SDS. Duration of hybridization isgenerally less than about 24 hours, usually about 4 to about 12 hours.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the T_(m) can be approximated fromthe equation of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284:T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M isthe molarity of monovalent cations, % GC is the percentage of guanosineand cytosine nucleotides in the DNA, % form is the percentage offormamide in the hybridization solution, and L is the length of thehybrid in base pairs. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of a complementary target sequencehybridizes to a perfectly matched probe. T_(m) is reduced by about 1° C.for each 1% of mismatching; thus, T_(m), hybridization, and/or washconditions can be adjusted to hybridize to sequences of the desiredidentity. For example, if sequences with ≧90% identity are sought, theT_(m) can be decreased 10° C. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point (T_(m))for the specific sequence and its complement at a defined ionic strengthand pH. However, severely stringent conditions can utilize ahybridization and/or wash at 1, 2, 3, or 4° C. lower than the thermalmelting point (T_(m)); moderately stringent conditions can utilize ahybridization and/or wash at 6, 7, 8, 9, or 10° C. lower than thethermal melting point (T_(m)); low stringency conditions can utilize ahybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower thanthe thermal melting point (T_(m)). Using the equation, hybridization andwash compositions, and desired T_(m), those of ordinary skill willunderstand that variations in the stringency of hybridization and/orwash solutions are inherently described. If the desired degree ofmismatching results in a T_(m) of less than 45° C. (aqueous solution) or32° C. (formamide solution), it is preferred to increase the SSCconcentration so that a higher temperature can be used. An extensiveguide to the hybridization of nucleic acids is found in Tijssen (1993)Laboratory Techniques in Biochemistry and MolecularBiology-Hybridization with Nucleic Acid Probes, Part I, Chapter 2(Elsevier, New York); and Ausubel et al., eds. (1995) Current Protocolsin Molecular Biology, Chapter 2 (Greene Publishing andWiley-Interscience, New York). See Sambrook et al. (1989) MolecularCloning: A Laboratory Manual (2d ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.).

Isolated Proteins and Variants and Fragments Thereof

Delta-endotoxin proteins are also encompassed within the presentinvention. By “delta-endotoxin protein” is intended a protein having theamino acid sequence set forth in SEQ ID NO:4, 5, 6, 13, or 14.Fragments, biologically active portions, and variants thereof are alsoprovided, and may be used to practice the methods of the presentinvention.

“Fragments” or “biologically active portions” include polypeptidefragments comprising amino acid sequences sufficiently identical to theamino acid sequence set forth in any of SEQ ID NO:4, 5, 6, 13, or 14 andthat exhibit insecticidal activity. A biologically active portion of adelta-endotoxin protein can be a polypeptide that is, for example, 10,25, 50, 100 or more amino acids in length. Such biologically activeportions can be prepared by recombinant techniques and evaluated forinsecticidal activity. Methods for measuring insecticidal activity arewell known in the art. See, for example, Czapla and Lang (1990) J. Econ.Entomol. 83:2480-2485; Andrews et al. (1988) Biochem. J. 252:199-206;Marrone et al. (1985) J. of Economic Entomology 78:290-293; and U.S.Pat. No. 5,743,477, all of which are herein incorporated by reference intheir entirety. As used here, a fragment comprises at least 8 contiguousamino acids of SEQ ID NO:4, 5, 6, 13, or 14. The invention encompassesother fragments, however, such as any fragment in the protein greaterthan about 10, 20, 30, 50, 100, 150, 200, 250, 300, 350, 400, 400, 450,500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100,1150, 1200, 1250, or 1300 amino acids.

By “variants” is intended proteins or polypeptides having an amino acidsequence that is at least about 60%, 65%, about 70%, 75%, about 80%,85%, about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identicalto the amino acid sequence of any of SEQ ID NO:4, 5, 6, 13, or 14.Variants also include polypeptides encoded by a nucleic acid moleculethat hybridizes to the nucleic acid molecule of SEQ ID NO:1, 2, 3, 11,or 12, or a complement thereof, under stringent conditions. Variantsinclude polypeptides that differ in amino acid sequence due tomutagenesis. Variant proteins encompassed by the present invention arebiologically active, that is they continue to possess the desiredbiological activity of the native protein, that is, retaininginsecticidal activity. Methods for measuring insecticidal activity arewell known in the art. See, for example, Czapla and Lang (1990) J. Econ.Entomol. 83:2480-2485; Andrews et al. (1988) Biochem. J. 252:199-206;Marrone et al. (1985) J. of Economic Entomology 78:290-293; and U.S.Pat. No. 5,743,477, all of which are herein incorporated by reference intheir entirety.

Bacterial genes, such as the axmi genes of this invention, quite oftenpossess multiple methionine initiation codons in proximity to the startof the open reading frame. Often, translation initiation at one or moreof these start codons will lead to generation of a functional protein.These start codons can include ATG codons. However, bacteria such asBacillus sp. also recognize the codon GTG as a start codon, and proteinsthat initiate translation at GTG codons contain a methionine at thefirst amino acid. Furthermore, it is not often determined a priori whichof these codons are used naturally in the bacterium. Thus, it isunderstood that use of one of the alternate methionine codons may alsolead to generation of delta-endotoxin proteins that encode insecticidalactivity. These delta-endotoxin proteins are encompassed in the presentinvention and may be used in the methods of the present invention.

Antibodies to the polypeptides of the present invention, or to variantsor fragments thereof, are also encompassed. Methods for producingantibodies are well known in the art (see, for example, Harlow and Lane(1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.; U.S. Pat. No. 4,196,265).

Altered or Improved Variants

It is recognized that DNA sequences of a delta-endotoxin may be alteredby various methods, and that these alterations may result in DNAsequences encoding proteins with amino acid sequences different thanthat encoded by a delta-endotoxin of the present invention. This proteinmay be altered in various ways including amino acid substitutions,deletions, truncations, and insertions of one or more amino acids of SEQID NO:4, 5, 6, 13, or 14, including up to about 2, about 3, about 4,about 5, about 6, about 7, about 8, about 9, about 10, about 15, about20, about 25, about 30, about 35, about 40, about 45, about 50, about55, about 60, about 65, about 70, about 75, about 80, about 85, about90, about 100, about 105, about 110, about 115, about 120, about 125,about 130 or more amino acid substitutions, deletions or insertions.

Methods for such manipulations are generally known in the art. Forexample, amino acid sequence variants of a delta-endotoxin protein canbe prepared by mutations in the DNA. This may also be accomplished byone of several forms of mutagenesis and/or in directed evolution. Insome aspects, the changes encoded in the amino acid sequence will notsubstantially affect the function of the protein. Such variants willpossess the desired insecticidal activity. However, it is understoodthat the ability of a delta-endotoxin to confer insecticidal activitymay be improved by the use of such techniques upon the compositions ofthis invention. For example, one may express a delta-endotoxin in hostcells that exhibit high rates of base misincorporation during DNAreplication, such as XL-1 Red (Stratagene). After propagation in suchstrains, one can isolate the delta-endotoxin DNA (for example bypreparing plasmid DNA, or by amplifying by PCR and cloning the resultingPCR fragment into a vector), culture the delta-endotoxin mutations in anon-mutagenic strain, and identify mutated delta-endotoxin genes withinsecticidal activity, for example by performing an assay to test forinsecticidal activity. Generally, the protein is mixed and used infeeding assays. See, for example Marrone et al. (1985) J. of EconomicEntomology 78:290-293. Such assays can include contacting plants withone or more insects and determining the plant's ability to surviveand/or cause the death of the insects. Examples of mutations that resultin increased toxicity are found in Schnepf et al. (1998) Microbiol. Mol.Biol. Rev. 62:775-806.

Alternatively, alterations may be made to the protein sequence of manyproteins at the amino or carboxy terminus without substantiallyaffecting activity. This can include insertions, deletions, oralterations introduced by modern molecular methods, such as PCR,including PCR amplifications that alter or extend the protein codingsequence by virtue of inclusion of amino acid encoding sequences in theoligonucleotides utilized in the PCR amplification. Alternatively, theprotein sequences added can include entire protein-coding sequences,such as those used commonly in the art to generate protein fusions. Suchfusion proteins are often used to (1) increase expression of a proteinof interest (2) introduce a binding domain, enzymatic activity, orepitope to facilitate either protein purification, protein detection, orother experimental uses known in the art (3) target secretion ortranslation of a protein to a subcellular organelle, such as theperiplasmic space of Gram-negative bacteria, or the endoplasmicreticulum of eukaryotic cells, the latter of which often results inglycosylation of the protein.

Variant nucleotide and amino acid sequences of the present inventionalso encompass sequences derived from mutagenic and recombinogenicprocedures such as DNA shuffling. With such a procedure, one or moredifferent delta-endotoxin protein coding regions can be used to create anew delta-endotoxin protein possessing the desired properties. In thismanner, libraries of recombinant polynucleotides are generated from apopulation of related sequence polynucleotides comprising sequenceregions that have substantial sequence identity and can be homologouslyrecombined in vitro or in vivo. For example, using this approach,sequence motifs encoding a domain of interest may be shuffled between adelta-endotoxin gene of the invention and other known delta-endotoxingenes to obtain a new gene coding for a protein with an improvedproperty of interest, such as an increased insecticidal activity.Strategies for such DNA shuffling are known in the art. See, forexample, Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751;Stemmer (1994) Nature 370:389-391; Crameri et al. (1997) Nature Biotech.15:436-438; Moore et al. (1997) J. Mol. Biol. 272:336-347; Zhang et al.(1997) Proc. Natl. Acad. Sci. USA 94:4504-4509; Crameri et al. (1998)Nature 391:288-291; and U.S. Pat. Nos. 5,605,793 and 5,837,458.

Domain swapping or shuffling is another mechanism for generating altereddelta-endotoxin proteins. Domains II and III may be swapped betweendelta-endotoxin proteins, resulting in hybrid or chimeric toxins withimproved insecticidal activity or target spectrum. Methods forgenerating recombinant proteins and testing them for insecticidalactivity are well known in the art (see, for example, Naimov et al.(2001) Appl. Environ. Microbiol. 67:5328-5330; de Maagd et al. (1996)Appl. Environ. Microbiol. 62:1537-1543; Ge et al. (1991) J. Biol. Chem.266:17954-17958; Schnepf et al. (1990) J. Biol. Chem. 265:20923-20930;Rang et al. 91999) Appl. Environ. Microbiol. 65:2918-2925).

Vectors

A delta-endotoxin sequence of the invention may be provided in anexpression cassette for expression in a plant of interest. By “plantexpression cassette” is intended a DNA construct that is capable ofresulting in the expression of a protein from an open reading frame in aplant cell. Typically these contain a promoter and a coding sequence.Often, such constructs will also contain a 3′ untranslated region. Suchconstructs may contain a “signal sequence” or “leader sequence” tofacilitate co-translational or post-translational transport of thepeptide to certain intracellular structures such as the chloroplast (orother plastid), endoplasmic reticulum, or Golgi apparatus.

By “signal sequence” is intended a sequence that is known or suspectedto result in cotranslational or post-translational peptide transportacross the cell membrane. In eukaryotes, this typically involvessecretion into the Golgi apparatus, with some resulting glycosylation.By “leader sequence” is intended any sequence that when translated,results in an amino acid sequence sufficient to trigger co-translationaltransport of the peptide chain to a sub-cellular organelle. Thus, thisincludes leader sequences targeting transport and/or glycosylation bypassage into the endoplasmic reticulum, passage to vacuoles, plastidsincluding chloroplasts, mitochondria, and the like.

By “plant transformation vector” is intended a DNA molecule that isnecessary for efficient transformation of a plant cell. Such a moleculemay consist of one or more plant expression cassettes, and may beorganized into more than one “vector” DNA molecule. For example, binaryvectors are plant transformation vectors that utilize two non-contiguousDNA vectors to encode all requisite cis- and trans-acting functions fortransformation of plant cells (Hellens and Mullineaux (2000) Trends inPlant Science 5:446-451). “Vector” refers to a nucleic acid constructdesigned for transfer between different host cells. “Expression vector”refers to a vector that has the ability to incorporate, integrate andexpress heterologous DNA sequences or fragments in a foreign cell. Thecassette will include 5′ and 3′ regulatory sequences operably linked toa sequence of the invention. By “operably linked” is intended afunctional linkage between a promoter and a second sequence, wherein thepromoter sequence initiates and mediates transcription of the DNAsequence corresponding to the second sequence. Generally, operablylinked means that the nucleic acid sequences being linked are contiguousand, where necessary to join two protein coding regions, contiguous andin the same reading frame. The cassette may additionally contain atleast one additional gene to be cotransformed into the organism.Alternatively, the additional gene(s) can be provided on multipleexpression cassettes.

“Promoter” refers to a nucleic acid sequence that functions to directtranscription of a downstream coding sequence. The promoter togetherwith other transcriptional and translational regulatory nucleic acidsequences (also termed “control sequences”) are necessary for theexpression of a DNA sequence of interest.

Such an expression cassette is provided with a plurality of restrictionsites for insertion of the delta-endotoxin sequence to be under thetranscriptional regulation of the regulatory regions.

The expression cassette will include in the 5′-3′ direction oftranscription, a transcriptional and translational initiation region(i.e., a promoter), a DNA sequence of the invention, and a translationaland transcriptional termination region (i.e., termination region)functional in plants. The promoter may be native, or analogous, orforeign or heterologous, to the plant host and/or to the DNA sequence ofthe invention. Additionally, the promoter may be the natural sequence oralternatively a synthetic sequence. Where the promoter is “native” or“homologous” to the plant host, it is intended that the promoter isfound in the native plant into which the promoter is introduced. Wherethe promoter is “foreign” or “heterologous” to the DNA sequence of theinvention, it is intended that the promoter is not the native ornaturally occurring promoter for the operably linked DNA sequence of theinvention.

The termination region may be native with the transcriptional initiationregion, may be native with the operably linked DNA sequence of interest,may be native with the plant host, or may be derived from another source(i.e., foreign or heterologous to the promoter, the DNA sequence ofinterest, the plant host, or any combination thereof). Convenienttermination regions are available from the Ti-plasmid of A. tumefaciens,such as the octopine synthase and nopaline synthase termination regions.See also Guerineau et al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot(1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149;Mogen et al. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene91:151-158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; andJoshi et al. (1987) Nucleic Acid Res. 15:9627-9639.

Where appropriate, the gene(s) may be optimized for increased expressionin the transformed host cell. That is, the genes can be synthesizedusing host cell-preferred codons for improved expression, or may besynthesized using codons at a host-preferred codon usage frequency.Generally, the GC content of the gene will be increased. See, forexample, Campbell and Gowri (1990) Plant Physiol. 92:1-11 for adiscussion of host-preferred codon usage. Methods are available in theart for synthesizing plant-preferred genes. See, for example, U.S. Pat.Nos. 5,380,831, and 5,436,391, and Murray et al. (1989) Nucleic AcidsRes. 17:477-498, herein incorporated by reference.

In one embodiment, the delta-endotoxin is targeted to the chloroplastfor expression. In this manner, where the delta-endotoxin is notdirectly inserted into the chloroplast, the expression cassette willadditionally contain a nucleic acid encoding a transit peptide to directthe delta-endotoxin to the chloroplasts. Such transit peptides are knownin the art. See, for example, Von Heijne et al. (1991) Plant Mol. Biol.Rep. 9:104-126; Clark et al. (1989) J. Biol. Chem. 264:17544-17550;Della-Cioppa et al. (1987) Plant Physiol. 84:965-968; Romer et al.(1993) Biochem. Biophys. Res. Commun. 196:1414-1421; and Shah et al.(1986) Science 233:478-481.

The delta-endotoxin gene to be targeted to the chloroplast may beoptimized for expression in the chloroplast to account for differencesin codon usage between the plant nucleus and this organelle. In thismanner, the nucleic acids of interest may be synthesized usingchloroplast-preferred codons. See, for example, U.S. Pat. No. 5,380,831,herein incorporated by reference.

Plant Transformation

Methods of the invention involve introducing a nucleotide construct intoa plant. By “introducing” is intended to present to the plant thenucleotide construct in such a manner that the construct gains access tothe interior of a cell of the plant. The methods of the invention do notrequire that a particular method for introducing a nucleotide constructto a plant is used, only that the nucleotide construct gains access tothe interior of at least one cell of the plant. Methods for introducingnucleotide constructs into plants are known in the art including, butnot limited to, stable transformation methods, transient transformationmethods, and virus-mediated methods.

By “plant” is intended whole plants, plant organs (e.g., leaves, stems,roots, etc.), seeds, plant cells, propagules, embryos and progeny of thesame. Plant cells can be differentiated or undifferentiated (e.g.callus, suspension culture cells, protoplasts, leaf cells, root cells,phloem cells, pollen).

“Transgenic plants” or “transformed plants” or “stably transformed”plants or cells or tissues refers to plants that have incorporated orintegrated exogenous nucleic acid sequences or DNA fragments into theplant cell. These nucleic acid sequences include those that areexogenous, or not present in the untransformed plant cell, as well asthose that may be endogenous, or present in the untransformed plantcell. “Heterologous” generally refers to the nucleic acid sequences thatare not endogenous to the cell or part of the native genome in whichthey are present, and have been added to the cell by infection,transfection, microinjection, electroporation, microprojection, or thelike.

Transformation of plant cells can be accomplished by one of severaltechniques known in the art. The delta-endotoxin gene of the inventionmay be modified to obtain or enhance expression in plant cells.Typically a construct that expresses such a protein would contain apromoter to drive transcription of the gene, as well as a 3′untranslated region to allow transcription termination andpolyadenylation. The organization of such constructs is well known inthe art. In some instances, it may be useful to engineer the gene suchthat the resulting peptide is secreted, or otherwise targeted within theplant cell. For example, the gene can be engineered to contain a signalpeptide to facilitate transfer of the peptide to the endoplasmicreticulum. It may also be preferable to engineer the plant expressioncassette to contain an intron, such that mRNA processing of the intronis required for expression.

Typically this “plant expression cassette” will be inserted into a“plant transformation vector”. This plant transformation vector may becomprised of one or more DNA vectors needed for achieving planttransformation. For example, it is a common practice in the art toutilize plant transformation vectors that are comprised of more than onecontiguous DNA segment. These vectors are often referred to in the artas “binary vectors”. Binary vectors as well as vectors with helperplasmids are most often used for Agrobacterium-mediated transformation,where the size and complexity of DNA segments needed to achieveefficient transformation is quite large, and it is advantageous toseparate functions onto separate DNA molecules. Binary vectors typicallycontain a plasmid vector that contains the cis-acting sequences requiredfor T-DNA transfer (such as left border and right border), a selectablemarker that is engineered to be capable of expression in a plant cell,and a “gene of interest” (a gene engineered to be capable of expressionin a plant cell for which generation of transgenic plants is desired).Also present on this plasmid vector are sequences required for bacterialreplication. The cis-acting sequences are arranged in a fashion to allowefficient transfer into plant cells and expression therein. For example,the selectable marker gene and the delta-endotoxin are located betweenthe left and right borders. Often a second plasmid vector contains thetrans-acting factors that mediate T-DNA transfer from Agrobacterium toplant cells. This plasmid often contains the virulence functions (Virgenes) that allow infection of plant cells by Agrobacterium, andtransfer of DNA by cleavage at border sequences and vir-mediated DNAtransfer, as is understood in the art (Hellens and Mullineaux (2000)Trends in Plant Science 5:446-451). Several types of Agrobacteriumstrains (e.g. LBA4404, GV3101, EHA101, EHA105, etc.) can be used forplant transformation. The second plasmid vector is not necessary fortransforming the plants by other methods such as microprojection,microinjection, electroporation, polyethylene glycol, etc.

In general, plant transformation methods involve transferringheterologous DNA into target plant cells (e.g. immature or matureembryos, suspension cultures, undifferentiated callus, protoplasts,etc.), followed by applying a maximum threshold level of appropriateselection (depending on the selectable marker gene) to recover thetransformed plant cells from a group of untransformed cell mass.Explants are typically transferred to a fresh supply of the same mediumand cultured routinely. Subsequently, the transformed cells aredifferentiated into shoots after placing on regeneration mediumsupplemented with a maximum threshold level of selecting agent. Theshoots are then transferred to a selective rooting medium for recoveringrooted shoot or plantlet. The transgenic plantlet then grows into amature plant and produces fertile seeds (e.g. Hiei et al. (1994) ThePlant Journal 6:271-282; Ishida et al. (1996) Nature Biotechnology14:745-750). Explants are typically transferred to a fresh supply of thesame medium and cultured routinely. A general description of thetechniques and methods for generating transgenic plants are found inAyres and Park (1994) Critical Reviews in Plant Science 13:219-239 andBommineni and Jauhar (1997) Maydica 42:107-120. Since the transformedmaterial contains many cells; both transformed and non-transformed cellsare present in any piece of subjected target callus or tissue or groupof cells. The ability to kill non-transformed cells and allowtransformed cells to proliferate results in transformed plant cultures.Often, the ability to remove non-transformed cells is a limitation torapid recovery of transformed plant cells and successful generation oftransgenic plants.

Transformation protocols as well as protocols for introducing nucleotidesequences into plants may vary depending on the type of plant or plantcell, i.e., monocot or dicot, targeted for transformation. Generation oftransgenic plants may be performed by one of several methods, including,but not limited to, microinjection, electroporation, direct genetransfer, introduction of heterologous DNA by Agrobacterium into plantcells (Agrobacterium-mediated transformation), bombardment of plantcells with heterologous foreign DNA adhered to particles, ballisticparticle acceleration, aerosol beam transformation (U.S. PublishedApplication No. 20010026941; U.S. Pat. No. 4,945,050; InternationalPublication No. WO 91/00915; U.S. Published Application No. 2002015066),Lec1 transformation, and various other non-particle direct-mediatedmethods to transfer DNA.

Methods for transformation of chloroplasts are known in the art. See,for example, Svab et al. (1990) Proc. Natl. Acad. Sci. USA 87:8526-8530;Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA 90:913-917; Svab andMaliga (1993) EMBO J. 12:601-606. The method relies on particle gundelivery of DNA containing a selectable marker and targeting of the DNAto the plastid genome through homologous recombination. Additionally,plastid transformation can be accomplished by transactivation of asilent plastid-borne transgene by tissue-preferred expression of anuclear-encoded and plastid-directed RNA polymerase. Such a system hasbeen reported in McBride et al. (1994) Proc. Natl. Acad. Sci. USA91:7301-7305.

Following integration of heterologous foreign DNA into plant cells, onethen applies a maximum threshold level of appropriate selection in themedium to kill the untransformed cells and separate and proliferate theputatively transformed cells that survive from this selection treatmentby transferring regularly to a fresh medium. By continuous passage andchallenge with appropriate selection, one identifies and proliferatesthe cells that are transformed with the plasmid vector. Molecular andbiochemical methods can then be used to confirm the presence of theintegrated heterologous gene of interest into the genome of thetransgenic plant.

The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick et al.(1986) Plant Cell Reports 5:81-84. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting hybrid having constitutive expression of the desiredphenotypic characteristic identified. Two or more generations may begrown to ensure that expression of the desired phenotypic characteristicis stably maintained and inherited and then seeds harvested to ensureexpression of the desired phenotypic characteristic has been achieved.In this manner, the present invention provides transformed seed (alsoreferred to as “transgenic seed”) having a nucleotide construct of theinvention, for example, an expression cassette of the invention, stablyincorporated into their genome.

Evaluation of Plant Transformation

Following introduction of heterologous foreign DNA into plant cells, thetransformation or integration of heterologous gene in the plant genomeis confirmed by various methods such as analysis of nucleic acids,proteins and metabolites associated with the integrated gene.

PCR analysis is a rapid method to screen transformed cells, tissue orshoots for the presence of incorporated gene at the earlier stage beforetransplanting into the soil (Sambrook and Russell (2001) MolecularCloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.). PCR is carried out using oligonucleotide primersspecific to the gene of interest or Agrobacterium vector background,etc.

Plant transformation may be confirmed by Southern blot analysis ofgenomic DNA (Sambrook and Russell, 2001, supra). In general, total DNAis extracted from the transformant, digested with appropriaterestriction enzymes, resolved in an agarose gel and transferred to anitrocellulose or nylon membrane. The membrane or “blot” is then probedwith, for example, radiolabeled ³²P target DNA fragment to confirm theintegration of introduced gene into the plant genome according tostandard techniques (Sambrook and Russell, 2001, supra).

In Northern blot analysis, RNA is isolated from specific tissues oftransformant, fractionated in a formaldehyde agarose gel, and blottedonto a nylon filter according to standard procedures that are routinelyused in the art (Sambrook and Russell, 2001, supra). Expression of RNAencoded by the delta-endotoxin is then tested by hybridizing the filterto a radioactive probe derived from a delta-endotoxin, by methods knownin the art (Sambrook and Russell, 2001, supra).

Western blot, biochemical assays and the like may be carried out on thetransgenic plants to confirm the presence of protein encoded by thedelta-endotoxin gene by standard procedures (Sambrook and Russell, 2001,supra) using antibodies that bind to one or more epitopes present on thedelta-endotoxin protein.

Insecticidal Activity in Plants

In another aspect of the invention, one may generate transgenic plantsexpressing a delta-endotoxin that has insecticidal activity. Methodsdescribed above by way of example may be utilized to generate transgenicplants, but the manner in which the transgenic plant cells are generatedis not critical to this invention. Methods known or described in the artsuch as Agrobacterium-mediated transformation, biolistic transformation,and non-particle-mediated methods may be used at the discretion of theexperimenter. Plants expressing a delta-endotoxin may be isolated bycommon methods described in the art, for example by transformation ofcallus, selection of transformed callus, and regeneration of fertileplants from such transgenic callus. In such process, one may use anygene as a selectable marker so long as its expression in plant cellsconfers ability to identify or select for transformed cells.

A number of markers have been developed for use with plant cells, suchas resistance to chloramphenicol, the aminoglycoside G418, hygromycin,or the like. Other genes that encode a product involved in chloroplastmetabolism may also be used as selectable markers. For example, genesthat provide resistance to plant herbicides such as glyphosate,bromoxynil, or imidazolinone may find particular use. Such genes havebeen reported (Stalker et al. (1985) J. Biol. Chem. 263:6310-6314(bromoxynil resistance nitrilase gene); and Sathasivan et al. (1990)Nucl. Acids Res. 18:2188 (AHAS imidazolinone resistance gene).Additionally, the genes disclosed herein are useful as markers to assesstransformation of bacterial or plant cells. Methods for detecting thepresence of a transgene in a plant, plant organ (e.g., leaves, stems,roots, etc.), seed, plant cell, propagule, embryo or progeny of the sameare well known in the art. In one embodiment, the presence of thetransgene is detected by testing for insecticidal activity.

Fertile plants expressing a delta-endotoxin may be tested forinsecticidal activity, and the plants showing optimal activity selectedfor further breeding. Methods are available in the art to assay forinsect activity. Generally, the protein is mixed and used in feedingassays. See, for example Marrone et al. (1985) J. of Economic Entomology78:290-293.

The present invention may be used for transformation of any plantspecies, including, but not limited to, monocots and dicots. Examples ofplants of interest include, but are not limited to, corn (maize),sorghum, wheat, sunflower, tomato, crucifers, peppers, potato, cotton,rice, soybean, sugarbeet, sugarcane, tobacco, barley, and oilseed rape,Brassica sp., alfalfa, rye, millet, safflower, peanuts, sweet potato,cassaya, coffee, coconut, pineapple, citrus trees, cocoa, tea, banana,avocado, fig, guava, mango, olive, papaya, cashew, macadamia, almond,oats, vegetables, ornamentals, and conifers. Vegetables include, but arenot limited to, tomatoes, lettuce, green beans, lima beans, peas, andmembers of the genus Curcumis such as cucumber, cantaloupe, and muskmelon. Ornamentals include, but are not limited to, azalea, hydrangea,hibiscus, roses, tulips, daffodils, petunias, carnation, poinsettia, andchrysanthemum. Preferably, plants of the present invention are cropplants (for example, maize, sorghum, wheat, sunflower, tomato,crucifers, peppers, potato, cotton, rice, soybean, sugarbeet, sugarcane,tobacco, barley, oilseed rape, etc.).

Use in Insect Control

General methods for employing strains comprising a nucleotide sequenceof the present invention, or a variant thereof, in insect control or inengineering other organisms as insecticidal agents are known in the art.See, for example U.S. Pat. No. 5,039,523 and EP 0480762A2.

The Bacillus strains containing a nucleotide sequence of the presentinvention, or a variant thereof, or the microorganisms that have beengenetically altered to contain an insecticidal gene and protein may beused for protecting agricultural crops and products from insects. In oneaspect of the invention, whole, i.e., unlysed, cells of a toxin(insecticide)-producing organism are treated with reagents that prolongthe activity of the toxin produced in the cell when the cell is appliedto the environment of target insect(s).

Alternatively, the insecticide is produced by introducing adelta-endotoxin gene into a cellular host. Expression of thedelta-endotoxin gene results, directly or indirectly, in theintracellular production and maintenance of the insecticide. In oneaspect of this invention, these cells are then treated under conditionsthat prolong the activity of the toxin produced in the cell when thecell is applied to the environment of target insect(s). The resultingproduct retains the toxicity of the toxin. These naturally encapsulatedinsecticides may then be formulated in accordance with conventionaltechniques for application to the environment hosting a target insect,e.g., soil, water, and foliage of plants. See, for example EPA 0192319,and the references cited therein. Alternatively, one may formulate thecells expressing a gene of this invention such as to allow applicationof the resulting material as an insecticide.

Insecticidal Compositions

The active ingredients of the present invention are normally applied inthe form of compositions and can be applied to the crop area or plant tobe treated, simultaneously or in succession, with other compounds. Thesecompounds can be fertilizers, weed killers, cryoprotectants,surfactants, detergents, insecticidal soaps, dormant oils, polymers,and/or time-release or biodegradable carrier formulations that permitlong-term dosing of a target area following a single application of theformulation. They can also be selective herbicides, chemicalinsecticides, virucides, microbicides, amoebicides, pesticides,fungicides, bacteriocides, nematocides, molluscicides or mixtures ofseveral of these preparations, if desired, together with furtheragriculturally acceptable carriers, surfactants or application-promotingadjuvants customarily employed in the art of formulation. Suitablecarriers and adjuvants can be solid or liquid and correspond to thesubstances ordinarily employed in formulation technology, e.g. naturalor regenerated mineral substances, solvents, dispersants, wettingagents, tackifiers, binders or fertilizers. Likewise the formulationsmay be prepared into edible “baits” or fashioned into pest “traps” topermit feeding or ingestion by a target pest of the insecticidalformulation.

Methods of applying an active ingredient of the present invention or anagrochemical composition of the present invention that contains at leastone of the insecticidal proteins produced by the bacterial strains ofthe present invention include leaf application, seed coating and soilapplication. The number of applications and the rate of applicationdepend on the intensity of infestation by the corresponding insect.

The composition may be formulated as a powder, dust, pellet, granule,spray, emulsion, colloid, solution, or such like, and may be prepared bysuch conventional means as desiccation, lyophilization, homogenation,extraction, filtration, centrifugation, sedimentation, or concentrationof a culture of cells comprising the polypeptide. In all suchcompositions that contain at least one such insecticidal polypeptide,the polypeptide may be present in a concentration of from about 1% toabout 99% by weight.

Lepidopteran, coleopteran, or other insects may be killed or reduced innumbers in a given area by the methods of the invention, or may beprophylactically applied to an environmental area to prevent infestationby a susceptible insect. Preferably the insect ingests, or is contactedwith, an insecticidally-effective amount of the polypeptide. By“insecticidally-effective amount” is intended an amount of theinsecticide that is able to bring about death to at least one insect, orto noticeably reduce insect growth, feeding, or normal physiologicaldevelopment. This amount will vary depending on such factors as, forexample, the specific target insects to be controlled, the specificenvironment, location, plant, crop, or agricultural site to be treated,the environmental conditions, and the method, rate, concentration,stability, and quantity of application of the insecticidally-effectivepolypeptide composition. The formulations may also vary with respect toclimatic conditions, environmental considerations, and/or frequency ofapplication and/or severity of insect infestation.

The insecticide compositions described may be made by formulating eitherthe bacterial cell, crystal and/or spore suspension, or isolated proteincomponent with the desired agriculturally-acceptable carrier. Thecompositions may be formulated prior to administration in an appropriatemeans such as lyophilized, freeze-dried, desiccated, or in an aqueouscarrier, medium or suitable diluent, such as saline or other buffer. Theformulated compositions may be in the form of a dust or granularmaterial, or a suspension in oil (vegetable or mineral), or water oroil/water emulsions, or as a wettable powder, or in combination with anyother carrier material suitable for agricultural application. Suitableagricultural carriers can be solid or liquid and are well known in theart. The term “agriculturally-acceptable carrier” covers all adjuvants,inert components, dispersants, surfactants, tackifiers, binders, etc.that are ordinarily used in insecticide formulation technology; theseare well known to those skilled in insecticide formulation. Theformulations may be mixed with one or more solid or liquid adjuvants andprepared by various means, e.g., by homogeneously mixing, blendingand/or grinding the insecticidal composition with suitable adjuvantsusing conventional formulation techniques. Suitable formulations andapplication methods are described in U.S. Pat. No. 6,468,523, hereinincorporated by reference.

“Pest” includes but is not limited to, insects, fungi, bacteria,nematodes, mites, ticks, and the like. Insect pests include insectsselected from the orders Coleoptera, Diptera, Hymenoptera, Lepidoptera,Mallophaga, Homoptera, Hemiptera, Orthroptera, Thysanoptera, Dermaptera,Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., particularlyColeoptera, Lepidoptera, and Diptera.

The order Coleoptera includes the suborders Adephaga and Polyphaga.Suborder Adephaga includes the superfamilies Caraboidea and Gyrinoidea,while suborder Polyphaga includes the superfamilies Hydrophiloidea,Staphylinoidea, Cantharoidea, Cleroidea, Elateroidea, Dascilloidea,Dryopoidea, Byrrhoidea, Cucujoidea, Meloidea, Mordelloidea,Tenebrionoidea, Bostrichoidea, Scarabaeoidea, Cerambycoidea,Chrysomeloidea, and Curculionoidea. Superfamily Caraboidea includes thefamilies Cicindelidae, Carabidae, and Dytiscidae. Superfamily Gyrinoideaincludes the family Gyrimidae. Superfamily Hydrophiloidea includes thefamily Hydrophilidae. Superfamily Staphylinoidea includes the familiesSilphidae and Staphylimidae. Superfamily Cantharoidea includes thefamilies Cantharidae and Lampyridae. Superfamily Cleroidea includes thefamilies Cleridae and Dermestidae. Superfamily Elateroidea includes thefamilies Elateridae and Buprestidae. Superfamily Cucujoidea includes thefamily Coccinellidae. Superfamily Meloidea includes the family Meloidae.Superfamily Tenebrionoidea includes the family Tenebrionidae.Superfamily Scarabaeoidea includes the families Passalidae andScarabaeidae. Superfamily Cerambycoidea includes the familyCerambycidae. Superfamily Chrysomeloidea includes the familyChrysomelidae. Superfamily Curculionoidea includes the familiesCurculionidae and Scolytidae.

The order Diptera includes the Suborders Nematocera, Brachycera, andCyclorrhapha. Suborder Nematocera includes the families Tipulidae,Psychodidae, Culicidae, Ceratopogonidae, Chironomidae, Simuliidae,Bibionidae, and Cecidomyiidae. Suborder Brachycera includes the familiesStratiomyidae, Tabanidae, Therevidae, Asilidae, Mydidae, Bombyliidae,and Dolichopodidae. Suborder Cyclorrhapha includes the Divisions Aschizaand Aschiza. Division Aschiza includes the families Phoridae, Syrphidae,and Conopidae. Division Aschiza includes the Sections Acalyptratae andCalyptratae. Section Acalyptratae includes the families Otitidae,Tephritidae, Agromyzidae, and Drosophilidae. Section Calyptrataeincludes the families Hippoboscidae, Oestridae, Tachimidae,Anthomyiidae, Muscidae, Calliphoridae, and Sarcophagidae.

The order Lepidoptera includes the families Papilionidae, Pieridae,Lycaenidae, Nymphalidae, Danaidae, Satyridae, Hesperiidae, Sphingidae,Saturniidae, Geometridae, Arctiidae, Noctuidae, Lymantriidae, Sesiidae,Crambidae, and Tineidae.

Nematodes include parasitic nematodes such as root-knot, cyst, andlesion nematodes, including Heterodera spp., Meloidogyne spp., andGlobodera spp.; particularly members of the cyst nematodes, including,but not limited to, Heterodera glycines (soybean cyst nematode);Heterodera schachtii (beet cyst nematode); Heterodera avenae (cerealcyst nematode); and Globodera rostochiensis and Globodera pailida(potato cyst nematodes). Lesion nematodes include Pratylenchus spp.

Insect pests of the invention for the major crops include: Maize:Ostrinia nubilalis, European corn borer; Agrotis ipsilon, black cutworm;Helicoverpa zea, corn earworm; Spodoptera frugiperda, fall armyworm;Diatraea grandiosella, southwestern corn borer; Elasmopalpuslignosellus, lesser cornstalk borer; Diatraea saccharalis, surgarcaneborer; Diabrotica virgifera, western corn rootworm; Diabroticalongicornis barberi, northern corn rootworm; Diabrotica undecimpunctatahowardi, southern corn rootworm; Melanotus spp., wireworms; Cyclocephalaborealis, northern masked chafer (white grub); Cyclocephala immaculata,southern masked chafer (white grub); Popillia japonica, Japanese beetle;Chaetocnema pulicaria, corn flea beetle; Sphenophorus maidis, maizebillbug; Rhopalosiphum maidis, corn leaf aphid; Anuraphis maidiradicis,corn root aphid; Blissus leucopterus leucopterus, chinch bug; Melanoplusfemurrubrum, redlegged grasshopper; Melanoplus sanguinipes, migratorygrasshopper; Hylemya platura, seedcorn maggot; Agromyza parvicornis,corn blot leafminer; Anaphothrips obscrurus, grass thrips; Solenopsismilesta, thief ant; Tetranychus urticae, twospotted spider mite;Sorghum: Chilo partellus, sorghum borer; Spodoptera frugiperda, fallarmyworm; Helicoverpa zea, corn earworm; Elasmopalpus lignosellus,lesser cornstalk borer; Feltia subterranea, granulate cutworm;Phyllophaga crinita, white grub; Eleodes, Conoderus, and Aeolus spp.,wireworms; Oulema melanopus, cereal leaf beetle; Chaetocnema pulicaria,corn flea beetle; Sphenophorus maidis, maize billbug; Rhopalosiphummaidis; corn leaf aphid; Sipha flava, yellow sugarcane aphid; Blissusleucopterus leucopterus, chinch bug; Contarinia sorghicola, sorghummidge; Tetranychus cinnabarinus, carmine spider mite; Tetranychusurticae, twospotted spider mite; Wheat: Pseudaletia unipunctata, armyworm; Spodoptera frugiperda, fall armyworm; Elasmopalpus lignosellus,lesser cornstalk borer; Agrotis orthogonia, western cutworm;Elasmopalpus lignosellus, lesser cornstalk borer; Oulema melanopus,cereal leaf beetle; Hypera punctata, clover leaf weevil; Diabroticaundecimpunctata howardi, southern corn rootworm; Russian wheat aphid;Schizaphis graminum, greenbug; Macrosiphum avenae, English grain aphid;Melanoplus femurrubrum, redlegged grasshopper; Melanoplusdifferentialis, differential grasshopper; Melanoplus sanguinipes,migratory grasshopper; Mayetiola destructor, Hessian fly; Sitodiplosismosellana, wheat midge; Meromyza americana, wheat stem maggot; Hylemyacoarctata, wheat bulb fly; Frankliniella fusca, tobacco thrips; Cephuscinctus, wheat stem sawfly; Aceria tulipae, wheat curl mite; Sunflower:Suleima helianthana, sunflower bud moth; Homoeosoma electellum,sunflower moth; zygogramma exclamationis, sunflower beetle; Bothyrusgibbosus, carrot beetle; Neolasioptera murtfeldtiana, sunflower seedmidge; Cotton: Heliothis virescens, cotton budworm; Helicoverpa zea,cotton bollworm; Spodoptera exigua, beet armyworm; Pectinophoragossypiella, pink bollworm; Anthonomus grandis, boll weevil; Aphisgossypii, cotton aphid; Pseudatomoscelis seriatus, cotton fleahopper;Trialeurodes abutilonea, bandedwinged whitefly; Lygus lineolaris,tarnished plant bug; Melanoplus femurrubrum, redlegged grasshopper;Melanoplus differentialis, differential grasshopper; Thrips tabaci,onion thrips; Franklinkiella fusca, tobacco thrips; Tetranychuscinnabarinus, carmine spider mite; Tetranychus urticae, twospottedspider mite; Rice: Diatraea saccharalis, sugarcane borer; Spodopterafrugiperda, fall armyworm; Helicoverpa zea, corn earworm; Colaspisbrunnea, grape colaspis; Lissorhoptrus oryzophilus, rice water weevil;Sitophilus oryzae, rice weevil; Nephotettix nigropictus, riceleafhopper; Blissus leucopterus leucopterus, chinch bug; Acrosternumhilare, green stink bug; Soybean: Pseudoplusia includens, soybeanlooper; Anticarsia gemmatalis, velvetbean caterpillar; Plathypenascabra, green cloverworm; Ostrinia nubilalis, European corn borer;Agrotis ipsilon, black cutworm; Spodoptera exigua, beet armyworm;Heliothis virescens, cotton budworm; Helicoverpa zea, cotton bollworm;Epilachna varivestis, Mexican bean beetle; Myzus persicae, green peachaphid; Empoasca fabae, potato leafhopper; Acrosternum hilare, greenstink bug; Melanoplus femurrubrum, redlegged grasshopper; Melanoplusdifferentialis, differential grasshopper; Hylemya platura, seedcornmaggot; Sericothrips variabilis, soybean thrips; Thrips tabaci, onionthrips; Tetranychus turkestani, strawberry spider mite; Tetranychusurticae, twospotted spider mite; Barley: Ostrinia nubilalis, Europeancorn borer; Agrotis ipsilon, black cutworm; Schizaphis graminum,greenbug; Blissus leucopterus leucopterus, chinch bug; Acrosternumhilare, green stink bug; Euschistus servus, brown stink bug; Deliaplatura, seedcorn maggot; Mayetiola destructor, Hessian fly; Petrobialatens, brown wheat mite; Oil Seed Rape: Brevicoryne brassicae, cabbageaphid; Phyllotreta cruciferae, Flea beetle; Mamestra configurata, Berthaarmyworm; Plutella xylostella, Diamond-back moth; Delia ssp., Rootmaggots.

Methods for Increasing Plant Yield

Methods for increasing plant yield are provided. The methods compriseintroducing into a plant or plant cell a polynucleotide comprising aninsecticidal sequence disclosed herein. As defined herein, the “yield”of the plant refers to the quality and/or quantity of biomass producedby the plant. By “biomass” is intended any measured plant product. Anincrease in biomass production is any improvement in the yield of themeasured plant product. Increasing plant yield has several commercialapplications. For example, increasing plant leaf biomass may increasethe yield of leafy vegetables for human or animal consumption.Additionally, increasing leaf biomass can be used to increase productionof plant-derived pharmaceutical or industrial products. An increase inyield can comprise any statistically significant increase including, butnot limited to, at least a 1% increase, at least a 3% increase, at leasta 5% increase, at least a 10% increase, at least a 20% increase, atleast a 30%, at least a 50%, at least a 70%, at least a 100% or agreater increase in yield compared to a plant not expressing theinsecticidal sequence.

In specific methods, plant yield is increased as a result of improvedinsect resistance of a plant expressing an insecticidal proteindisclosed herein. Expression of the insecticidal protein results in areduced ability of an insect to infest or feed on the plant, thusimproving plant yield.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL Example 1 Discovery of a Novel Toxin Gene Axmi115 from theBacillus thuringiensis Strain ATX12983

The complete gene sequence was identified from the selected strain viathe MiDAS genomics approach as follows:

-   -   Preparation of extrachromosomal DNA from the strain.        Extrachromosomal DNA contains a mixture of some or all of the        following: plasmids of various size; phage chromosomes; genomic        DNA fragments not separated by the purification protocol; other        uncharacterized extrachromosomal molecules.    -   Mechanical or enzymatic shearing of the extrachromosomal DNA to        generate size-distributed fragments.    -   Sequencing of the fragmented DNA    -   Identification of putative toxin genes via homology and/or other        computational analyses.    -   When required, sequence finishing of the gene of interest by one        of several PCR or cloning strategies (e.g. TAIL-PCR).        The novel gene is referred to herein as axmi-115 (SEQ ID NO:3),        and the encoded amino acid referred to as AXMI-115 (SEQ ID        NO:6). Synthetic nucleotide sequences encoding AXMI-115 is set        forth in SEQ ID NO:15 and 16.

Gene and Protein Characteristics

Gene length, DNA base pairs: 2409Protein length, amino acid residues: 803Estimated protein molecular weight, Da: 90877Known homologs and approximate percent identity:

Vip3Af1—70.7%

Axmi005—70.4%

Axmi026—70.4%

Vip3Aa7—70.1

Example 2 Novel Insecticidal Protein AXMI-005 from the Bacillusthuringiensis Strain ATX13002

The AXMI-005 insecticidal gene was identified from the strain ATX13002using the MiDAS approach as described in U.S. Patent Publication No.20040014091, which is herein incorporated by reference in its entirety,using the following steps:

Steps taken in the current strategy to gene discovery:Step 1: Culture from the strain was grown in large quantities. Theplasmid DNA was then separated from the chromosomal DNA by a cesiumchloride gradient spun in an ultracentrifuge. The purified plasmid DNAwas then nebulized to a 5-10 kb size range appropriate for coverage ofan average sized coding region. The fragment ends were polished thenligated overnight into a vector cut with a restriction enzyme producingblunt ends.Step 3: Once the shotgun library quality was checked and confirmed,colonies were grown, prepped and sequenced in a 96-well format. Thelibrary plates were end sequenced off of the vector backbone for initialscreening.Step 5: All of the reads were compiled into an assembly project andaligned together to form contigs. These contigs, along with anyindividual read that may not have been added to a contig, were analyzedusing BLAST, using a batch format, against an internal database made upof all classes of known delta-endotoxin genes. Any contigs or individualreads that pulled up any homology to a known gene were analyzed furtherby selecting a single clone from the library that covered the entirehypothesized coding region.Step 6: The individual clone covering the area of interest was thenwalked over read by read by designing primers to extend the sequence.This was done until both end reads of the clone were joined and thecoverage was at least 2×. The completed contig of the single clone wasthen analyzed using BLAST (both blastn and blastx) against a publicdatabase of all known insecticidal genes. Hits from both searches werethen pulled from an internal database of all the genes (clipped tocoding sequence only) and aligned with the completed library clonesequence to determine the percentage of divergence from the known gene.

A novel gene, referred to herein as axmi-005 (SEQ ID NO:1), and theencoded amino acid referred to as AXMI-005 (SEQ ID NO:4) were identifiedby this approach. Searching of public sequence databases, including theGENBANK® databases, showed that AXMI-005 is a unique protein that hashighest homology (94.9%) to the vip3Aa insecticidal protein (GenePept IDL48841).

A synthetic sequence encoding the AXMI-005 protein was designed andtermed optaxmi-005. The nucleotide sequence is set forth in SEQ ID NO:7and encodes the amino acid sequence set forth in SEQ ID NO:9 (with theaddition of a C-terminal histidine tag). The optaxmi-005 gene disclosedherein can be used with or without the C-terminal histidine tag.

Example 3 Discovery of a Novel Toxin Gene Axmi-113 from the Bacillusthuringiensis Strain ATX12987

The complete gene sequence was identified from the selected strain viathe MiDAS genomics approach as follows:

-   -   Preparation of extrachromosomal DNA from the strain.        Extrachromosomal DNA contains a mixture of some or all of the        following: plasmids of various size; phage chromosomes; genomic        DNA fragments not separated by the purification protocol; other        uncharacterized extrachromosomal molecules.    -   Mechanical or enzymatic shearing of the extrachromosomal DNA to        generate size-distributed fragments.    -   Sequencing of the fragmented DNA    -   Identification of putative toxin genes via homology and/or other        computational analyses.    -   When required, sequence finishing of the gene of interest by one        of several PCR or cloning strategies (e.g. TAIL-PCR).        The novel gene is referred to herein as axmi-113 (SEQ ID NO:2),        and the encoded amino acid referred to as AXMI-113 (SEQ ID        NO:5).

Gene and Protein Characteristics

Gene length, DNA base pairs: 2385Protein length, amino acid residues: 795Estimated protein molecular weight, Da: 89475Known homologs and approximate percent identity:

Vip3Ah—99%

Vip3Aa18—79.8%

Axmi005—79%

A synthetic sequence encoding the AXMI-113 protein was designed andtermed optaxmi-113. The nucleotide sequence is set forth in SEQ ID NO:8and encodes the amino acid sequence set forth in SEQ ID NO:5 or 10 (withthe addition of a C-terminal histidine tag). The optaxmi-113 genedisclosed herein can be used with or without the C-terminal histidinetag.

Example 4 Discovery of Novel Toxin Genes Axmi-163 and Axmi-184 from theBacillus thuringiensis Strain ATX14775

The complete gene sequence for each was identified from the selectedstrain via the MiDAS genomics approach as follows:

-   -   Preparation of extrachromosomal DNA from the strain.        Extrachromosomal DNA contains a mixture of some or all of the        following: plasmids of various size; phage chromosomes; genomic        DNA fragments not separated by the purification protocol; other        uncharacterized extrachromosomal molecules.    -   Mechanical or enzymatic shearing of the extrachromosomal DNA to        generate size-distributed fragments.    -   Sequencing of the fragmented DNA    -   Identification of putative toxin genes via homology and/or other        computational analyses.    -   When required, sequence finishing of the gene of interest by one        of several PCR or cloning strategies (e.g. TAIL-PCR).        The novel gene referred to herein as axmi-163 is set forth in        SEQ ID NO:11, and the encoded amino acid referred to as AXMI-163        is set forth in SEQ ID NO:13.

Gene and Protein Characteristics

Gene length, DNA base pairs: 2370Protein length, amino acid residues: 790Estimated protein molecular weight, Da: 88,700Known homologs and approximate percent identity:

SEQ ID NO:17 from U.S. Pat. No. 7,129,212—98%

Axmi005—78%

The novel gene referred to herein as axmi-184 is set forth in SEQ IDNO:12, and the encoded amino acid referred to as AXMI-184 is set forthin SEQ ID NO:14. Synthetic nucleotide sequences encoding AXMI-184 areset forth in SEQ ID NO:17 and 18.

Gene and Protein Characteristics

Gene length, DNA base pairs: 2370Protein length, amino acid residues: 790Estimated protein molecular weight, Da: 88,300Known homologs and approximate percent identity:

Vip3Af1—93%

Axmi005—86%

Example 5 Construction of Synthetic Sequences

In one aspect of the invention, synthetic axmi sequences are generated.These synthetic sequences have an altered DNA sequence relative to theparent axmi sequence, and encode a protein that is collinear with theparent AXMI protein to which it corresponds, but lacks the C-terminal“crystal domain” present in many delta-endotoxin proteins.

In another aspect of the invention, modified versions of synthetic genesare designed such that the resulting peptide is targeted to a plantorganelle, such as the endoplasmic reticulum or the apoplast. Peptidesequences known to result in targeting of fusion proteins to plantorganelles are known in the art. For example, the N-terminal region ofthe acid phosphatase gene from the White Lupin Lupinus albus (GenebankID GI:14276838; Miller et al. (2001) Plant Physiology 127: 594-606) isknown in the art to result in endoplasmic reticulum targeting ofheterologous proteins. If the resulting fusion protein also contains anendoplasmic retention sequence comprising the peptideN-terminus-lysine-aspartic acid-glutamic acid-leucine (i.e. the “KDEL”motif (SEQ ID NO:19) at the C-terminus, the fusion protein will betargeted to the endoplasmic reticulum. If the fusion protein lacks anendoplasmic reticulum targeting sequence at the C-terminus, the proteinwill be targeted to the endoplasmic reticulum, but will ultimately besequestered in the apoplast.

Example 6 Expression in Bacillus

As an example of the expression of the genes and proteins of theinvention in Bacillus species, the insecticidal gene disclosed herein isamplified by PCR, and the PCR product is cloned into the Bacillusexpression vector pAX916, or another suitable vector, by methods wellknown in the art. The resulting Bacillus strain, containing the vectorwith axmi gene is cultured on a conventional growth media, such as CYSmedia (10 g/l Bacto-casitone; 3 g/l yeast extract; 6 g/l KH₂PO₄; 14 g/lK₂HPO₄; 0.5 mM MgSO₄; 0.05 mM MnCl₂; 0.05 mM FeSO₄), until sporulationis evident by microscopic examination. Samples are prepared and testedfor activity in bioassays.

Example 7 Expression in E. coli

As an example of a method of expression of the genes and proteins of theinvention in E. coli based systems, the complete ORF of each axmi geneis cloned into an E. coli expression vector based on pRSF1b. Theresulting clones are confirmed by restriction analysis and finally, bycomplete sequencing of the cloned gene.

For expression in E. coli, BL21*DE3 is transformed with the vectorexpressing the axmi gene. Single colonies are inoculated in LBsupplemented with kanamycin and grown overnight at 37° C. The followingday, fresh medium is inoculated in duplicate with 1% of overnightculture and grown at 37° C. to logarithmic phase. Subsequently, culturesare induced with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 3hours at 37° C. or overnight at 20° C. Each cell pellet is suspended in50 mM sodium carbonate buffer, pH 10.5 supplemented with 1 mM DTTdithiothreitol and sonicated. Samples are prepared and tested foractivity in bioassays.

Example 8 Expression of AXMI-115, AXMI-113, and AXMI-005 in E. coli

E. coli clones were generated that contained DNA segments containing thecomplete open reading frame as well as a portion of the DNA regionnaturally occurring upstream and adjacent to each gene. This DNA segmentfor each of axmi-113 (SEQ ID:2), axmi-115 (SEQ ID NO:3) or axmi-005 (SEQID NO:1) was amplified and cloned into the vector pAX916, to yield theclones pAX5463, pAX5464 and pAX5465 respectively. The resulting cloneswere confirmed by restriction analysis and by complete sequencing of thecloned fragments.

E. coli cells were transformed with each of the clones pAX5463, pAX5464and pAX5465.

axmi005, axmi113 and axmi115 genes that had their codons optimized forexpression in corn, and had a C-terminal his6-tag added, were alsoexpressed from E. coli expression vector utilizing T7 promoter. Inaddition, constructs were also generated that expressed N-terminalhis6-tagged or untagged versions of optaxmi005 (pAX5475, pAX5478) andoptaxmi115 (pAX5476, pAX5477).

Single E. coli colonies of the axmi-115, axmi-113, and axmi-005expressing clones were then grown overnight at 37° C. in LB medium. Thefollowing day, fresh medium was inoculated in duplicate with 1% ofovernight culture and grown at 37° C. to logarithmic phase.Subsequently, cultures were induced with 1 mM IPTG overnight at 20° C.The resulting cells were collected by centrifugation, and suspended ineither 50 mM sodium carbonate buffer, pH 10.5 supplemented with 1 mM DTTor 50 mM Tris Cl buffer, pH 8 with 1 mM DTT prior to sonication.SDS-PAGE analysis showed expression of a ˜90 kD protein in all samples.

Example 9 Insect Bioassays of E. coli Expressed Proteins

Soluble extracts containing AXMI-005, AXMI-113, or AXMI-115 were testedin insect assays with appropriate controls. Twenty four well tissueculture plates (Corning) were filled with 1 ml of multi-species diet(Bio-Serv) and allowed to solidify. Once solidified, 40 μl of proteinsample was placed on the diet surface of each well and allowed to soakin/dry at room temperature. Depending upon the experiment, either eggmasses or neonate larvae were placed in each well. Plates were sealedwith gas-permeable membranes (Research Products International) andincubated at 25° C. and 90% relative humidity. After five or seven days,samples were scored visually compared to a buffer only ornon-transformed extract control.

Strong activity of AXMI-005 extracts was observed on Helicoverpa zea(HZ), Heliothis virescens (HV), Fall Armyworm (FAW), Black cutworm(BCW), Sugarcane borer (SCB), and Velvet Bean caterpillar (VBC).AXMI-005 also showed activity on Southwestern Corn Borer (SWCB).

Strong activity of AXMI-115 extracts was observed on Heliothisvirescens, Fall Armyworm, Black cutworm, and Velvet Bean caterpillar.AXMI-115 also exhibited activity on the European Corn Borer (ECB), SCB,SWCB, and Diamondback moth (DBM). Activity of AXMI-115 on Helicoverpazea (HZ) was less pronounced than for the other insects tested, but wasstill significant.

Activity of each of the AXMI-005 and AXMI-115 extracts was scored, andassigned a number from 1 to 5 based on relative activity in the assays.A summary of the scores in a particular assay is shown in Table 1.

AXMI-005 showed some activity on SWCB (score of 2) and high levels ofactivity on Hz, Hv, FAW, BCW and VBC (scores of 4 to 5). AXMI-115 showedhigh levels of activity on SWCB (80% mortality), ECB, FAW and VBC(scores of 4 to 5) and lesser activity on Hz and Hv. AXMI-113 alsoshowed high activity on SWCB (score of 4 with 20% mortality) and on SCB.No activity was seen on the other insects tested.

TABLE 1 Insecticidal activity of AXMI-115, AXMI-113, and AXMI-005*Axmi115 Axmi115 Axmi005 Axmi005 Axmi113 (pH 10.5) (pH 8) (pH10.5) (pH 8)(pH 10.5) Hz 0 0 4 4 0 ECB 2 4 0 0 0 live infest Hv 0 0 4 4/5 0 FAW 4 24/5 4/5 0 BCW 0 0 3/4 4 0 VBC 4 3 4 4/5 0 SWCB 4; 80% 3; 50% 2 ND 4; 20%mortality mortality mortality SCB ND 3; 25% ND 4; 100% 3; 50% mortalitymortality mortality DBM ND 2/3 0 0 0 *= represented as stunt andmortality percent where stunting is scored according to the followingscale: Score Definition 0 No Activity 1 Slight, non-uniform stunt 2Non-uniform stunt 3 Uniform stunt 4 Uniform stunt with mortality(expressed as a percentage) 5 Uniform stunt with 100% mortality

Example 10 Bioassay of Axmi184 Gene Expression and Purification

-   -   The DNA region encoding the toxin domain of Axmi184 was cloned        into an E. coli expression vector pMAL-C4× behind the malE gene        coding for Maltose binding protein (MBP). This in-frame fusion        resulted in MBP-Axmi084 fusion protein expression in E. coli.    -   For expression in E. coli, BL21*DE3 was transformed with        individual plasmids. Single colony was inoculated in LB        supplemented with carbenicillin and glucose, and grown overnight        at 37° C. The following day, fresh medium was inoculated with 1%        of overnight culture and grown at 37° C. to logarithmic phase.        Subsequently, cultures were induced with 0.3 mM IPTG for        overnight at 20° C. Each cell pellet was suspended in 20 mM        Tris-Cl buffer, pH 7.4+200 mM NaCl+1 mM DTT+protease inhibitors        and sonicated. Analysis by SDS-PAGE confirmed expression of        fusion proteins.    -   Total cell free extracts were run over amylose column attached        to FPLC for affinity purification of MBP-AXMI184 fusion        proteins. Bound fusion protein was eluted from the resin with 10        mM maltose solution. Purified fusion proteins were then cleaved        with either Factor Xa or trypsin to remove the amino terminal        MBP tag from the AXMI184 protein. Cleavage and solubility of the        proteins was determined by SDS-PAGE.    -   Cleaved proteins were tested in insect assays with appropriate        controls. A 5-day read of the plates showed following activities        of AXMI-184 against Diamondback moth.

Example 11 Domain Swapping

axmi005, axmi113 and axmi115 genes that had their codons optimized forexpression in corn were used in this example. Plasmids expressinguntagged versions of optaxmi005 (pAX5478), optaxmi113 (pAX5493) andoptaxmi115 (pAX5477) were used to design DNA swap constructs as describehere.

AXMI-005, AXMI-113 and AXMI-115 have significant sequenceidentity/similarity in their N-terminal ⅔^(rd) region. The remaining⅓^(rd) region in their C-termini (CT) shows substantial sequencedivergence as seen in the protein sequence alignment provided as FIGS.1A and 1B.

The protein region of AXMI-113 between the forward and reverse arrowsshown in FIG. 1 was replaced with the corresponding fragment of eitherAXMI-005 (to give pAX5492) or AXMI-115 (pAX5494).

For expression in E. coli, BL21*DE3 was transformed with individualconstructs. A single colony was inoculated in LB supplemented withkanamycin and grown overnight at 37° C. The following day, fresh mediumwas inoculated in duplicate with 1% of overnight culture and grown at37° C. to logarithmic phase. Subsequently, cultures were induced with 1mM IPTG overnight at 20° C. Cell pellet was suspended in 50 mM sodiumcarbonate buffer, pH 10.5 supplemented with 1 mM DTT, and sonicated.Analysis by SDS-PAGE showed extremely good soluble expression of allproteins.

Filter sterilized, soluble extracts expressing OptAxmi005, 113, 115,Optaxmi113+CT of Optaxmi005 and Optaxmi113+CT of Optaxmi115 were testedin insect assays with appropriate controls. As shown in Example 9,AXMI-113 showed high activity on SWCB (25% mortality). It showed anadditional activity on SCB (50% mortality).

Also as shown in Example 9, AXMI-005 showed activity on SWCB, Hz, Hv,FAW, BCW and VBC. It showed an additional activity on SCB (25%mortality). AXMI-115 was also found to have some activity on SCB.

The fusion of AXMI-113+CT of AXMI-005 showed all of the insectactivities seen with AXMI-005. In other words, replacement of theC-terminal fragment of AXMI-113 with that of AXMI-005 bestowed upon itthe insect activities that were otherwise missing in its naturallyoccurring form.

Additional toxin protein sequences can be generated by swapping domainsfrom one protein into another. For example, one or more of the AXMI-005domains shown in FIG. 2 are introduced into AXMI-115. The domains areintroduced using the sense (“s”) and antisense (“a”) oligonucleotidesshown in Table 2. The portion of the axmi-005 sequence that is beingintroduced into the axmi-115 sequence is shown in bold print. Theflanking sequences in each oligonucleotide are axmi-115 sequences thatare used for annealing the oligonucleotides to the axmi-115 template.The number following the term “sub” in each primer name corresponds tothe numbered boxes in FIG. 2. Similar olignonucleotides can be designedto swap domains between multiple sequences, for example, between theAXMI-005, AXMI-113, AXMI-115, AXMI-163 and AXMI-184 sequences describedherein.

TABLE 2  Oligonucleotide SEQ ID primer Sequence NO: axmi115sub1 sAAC ACC GGC GGC GTC AAT GGA ACA AGG 20 GCG CTC TTC ACC CA axmi115sub1 aTGG GTG AAG AGC GCC CTT GTT CCA TTG 21 ACG CCG CCG GTG TT axmi115sub10 sGCC CGG AGC TCA TCA ATG TCA ACA ACT 22GGA TCA GAA CTG GCA CCA CCT ACA TCA C axmi115sub10 aGTG ATG TAG GTG GTG CCA GTT CTG ATC 23CAG TTG TTG ACA TTG ATG AGC TCC GGG C axmi115sub11 sATG ATT GGG AGA GGT TCG GAA GCA CCC 24 ACA TCA GCG GCA ATG AGC TGA GGaxmi115sub11 a CCT CAG CTC ATT GCC GCT GAT GTG GGT 25GCT TCC GAA CCT CTC CCA ATC AT axmi115sub12 sCTA CAT CAC CGG CAA TAC CTT GAC GCT 26CTA CCA AGG AGG AGG AGG CTA CTT CCG C axmi115sub12 aGCG GAA GTA GCC TCC TCC TCC TTG GTA 27GAG CGT CAA GGT ATT GCC GGT GAT GTA G axmi115sub14 sCGA CAG CTA CAG CAC CTA CAG GGT GAA 28CTT CTC CGT CAC CGG CTG GGC CAA GGT GAT axmi115sub14 aATC ACC TTG GCC CAG CCG GTG ACG GAG 29AAG TTC ACC CTG TAG GTG CTG TAG CTG TCG axmi115sub15 sGCT TCA GCG GCC TCG ACG CCA ATG TGA 30 GGA TCA GAA ACA GCC GCG GCaxmi115sub15 a GCC GCG GCT GTT TCT GAT CCT CAC ATT 31GGC GTC GAG GCC GCT GAA GC axmi115sub16 sGTG AAG AAC AGC CGC GAG GTG CTC TTC 32GAG AAG AGA TAC ATG AAT GGA AGC AGC TAT GA axmi115sub16 aTCA TAG CTG CTT CCA TTC ATG TAT CTC 33TTC TCG AAG AGC ACC TCG CGG CTG TTC TTC AC axmi115sub17 sTTC GAG AAG GTG AAG AAC AGC GGC GCC 34AAG GAT GTT TCA GAG AGC TTC ACC ACC axmi115sub17 aGGT GGT GAA GCT CTC TGA AAC ATC CTT 35GGC GCC GCT GTT CTT CAC CTT CTC GAA axmi115sub19 sGCT TCT TCA TCG AGC TCA GCC AAG GCA 36ACA ACC TCT ATA GCA GCA CCT TCC AC axmi115sub19 aGTG GAA GGT GCT GCT ATA GAG GTT GTT 37GCC TTG GCT GAG CTC GAT GAA GAA GC axmi115sub2 sGAA GCA AGG CGC TCT ATG TTC ACA AGG 38 ATG GAG GCT TCA GCC AGT TCA TCGaxmi115sub2 a CGA TGA ACT GGC TGA AGC CTC CAT CCT 39TGT GAA CAT AGA GCG CCT TGC TTC axmi115sub20 sCCG CCG AGA GGA CAG GAG GGC CGC TGG 40 TGA AGT TCA GAG ACA TCA GCA TCaxmi115sub20 a GAT GCT GAT GTC TCT GAA CTT CAC CAG 41CGG CCC TCC TGT CCT CTC GGC GG axmi115sub21 sAGC ACC TTC CAC AGC TTC AAT GAT GTG 42 AGC ATC AAG TAA GGC GCG CCGaxmi115sub21 a CGG CGC GCC TTA CTT GAT GCT CAC ATC 43ATT GAA GCT GTG GAA GGT GCT axmi115sub3 sCGA CAA GCT AAA GCC CAA GAC AGA ATA 44 TGT CAT CCA GTA CAC CGT CAA Gaxmi115sub3 a CTT GAC GGT GTA CTG GAT GAC ATA TTC 45TGT CTT GGG CTT TAG CTT GTC G axmi115sub5 sCCT ACG AGG ACA CCA ATA ACA ACA ACC 46TGG AGG ACT ACC AAA CAA TTG CTG TGA AG axmi115sub5 aCTT CAC AGC AAT TGT TTG GTA GTC CTC 47CAG GTT GTT GTT ATT GGT GTC CTC GTA GG axmi115sub6 sGAG GAG TTC CAA ACA ATT ACC AAG AGG 48TTC ACC ACC GGC ACA GAT TTG AGC CAG ACC axmi115sub6 aGGT CTG GCT CAA ATC TGT GCC GGT GGT 49GAA CCT CTT GGT AAT TGT TTG GAA CTC CTC axmi115sub7 sCAC CTC AGA AAC AGA TTT GAA GGG CGT 50CTA CCT CAT CTT GAA GAG CCA AAA TGG ATA T axmi115sub7 aATA TCC ATT TTG GCT CTT CAA GAT GAG 51GTA GAC GCC CTT CAA ATC TGT TTC TGA GGT G axmi115sub9 sTCC TGG AGG CCA AGC CAT CAG AGA AGC 52 TGC TCA GCC CGG AGC TCAaxmi115sub9 a TGA GCT CCG GGC TGA GCA GCT TCT CTG 53ATG GCT TGG CCT CCA GGA axmi115sub13 sATC ATT CAA GAG GAG GCA ACC TCA AGC 54AGA ACC TCC AGC TTG ACA GCT TCA GCA CCT ACG ACC TCA G axmi115sub13 aCTG AGG TCG TAG GTG CTG AAG CTG TCA 55AGC TGG AGG TTC TGC TTG AGG TTG CCT CCT CTT GAA TGA T axmi115sub18 sGCT ATG AGG ACA TCT CAG AGA TCT TCA 56CCA CCA AGC TGG GCA AGG ACA ACT TC TAC A TCG AGC TCA CCG Caxmi115sub18 a GCG GTG AGC TCG AT GTA G AAG TTG TCC 57TTG CCC AGC TTG GTG GTG AAG ATC TCT GAG ATG TCC TCA TAG C axmi115sub4 sCAA GGG CAA GCC GTC AAT CCA CCT CAA 58 GAA TGA GAA CAC CGG CTA CAT CCACTAC GA GGA CAC CAA TGG axmi115sub4 aCCA TTG GTG TCC TCG TAG TGG ATG TAG 59CCG GTG TTC TCA TTC TTG AGG TGG ATT GAC GGC TTG CCC TTG axmi115sub8 sCAA GAG CCA AAA TGG AGA TGA AGC ATG 60GGG AGA CAA CTT CAC CAT CCT GGA GAT CTC GCT CTT CGA GAC ACC AGA Aaxmi115sub8 a TTC TGG TGT CTC GAA GAG CGA GAT CTC 61CAG GAT GGT GAA GTT GTC TCC CCA TGC TTC ATC TCC ATT TTG GCT CTT G

Example 12 Additional Assays for Pesticidal Activity

The ability of an insecticidal protein to act as a pesticide upon a pestis often assessed in a number of ways. One way well known in the art isto perform a feeding assay. In such a feeding assay, one exposes thepest to a sample containing either compounds to be tested, or controlsamples. Often this is performed by placing the material to be tested,or a suitable dilution of such material, onto a material that the pestwill ingest, such as an artificial diet. The material to be tested maybe in a liquid, solid, or slurry form. The material to be tested may beplaced upon the surface and then allowed to dry or incorporate into thediet. Alternatively, the material to be tested may be mixed with amolten artificial diet, then dispensed into the assay chamber. The assaychamber may be, for example, a cup, a dish, or a well of a microtiterplate.

Assays for sucking pests (for example aphids) may involve separating thetest material from the insect by a partition, ideally a portion that canbe pierced by the sucking mouth parts of the sucking insect, to allowingestion of the test material. Often the test material is mixed with afeeding stimulant, such as sucrose, to promote ingestion of the testcompound.

Other types of assays can include microinjection of the test materialinto the mouth, or gut of the pest, as well as development of transgenicplants, followed by test of the ability of the pest to feed upon thetransgenic plant. Plant testing may involve isolation of the plant partsnormally consumed, for example, small cages attached to a leaf, orisolation of entire plants in cages containing insects.

Other methods and approaches to assay pests are known in the art, andcan be found, for example in Robertson, J. L. & H. K. Preisler. 1992.Pesticide bioassays with arthropods. CRC, Boca Raton, Fla.Alternatively, assays are commonly described in the journals “ArthropodManagement Tests” and “Journal of Economic Entomology” or by discussionwith members of the Entomological Society of America (ESA).

Example 13 Vectoring of the Insecticidal Genes of the Invention forPlant Expression

Each of the coding regions of the genes of the invention is connectedindependently with appropriate promoter and terminator sequences forexpression in plants. Such sequences are well known in the art and mayinclude the rice actin promoter or maize ubiquitin promoter forexpression in monocots, the Arabidopsis UBQ3 promoter or CaMV 35Spromoter for expression in dicots, and the nos or PinIl terminators.Techniques for producing and confirming promoter—gene—terminatorconstructs also are well known in the art.

Example 14 Transformation of the Genes of the Invention into Plant Cellsby Agrobacterium-Mediated Transformation

Ears are collected 8-12 days after pollination. Embryos are isolatedfrom the ears, and those embryos 0.8-1.5 mm in size are used fortransformation. Embryos are plated scutellum side-up on a suitableincubation media, and incubated overnight at 25° C. in the dark.However, it is not necessary per se to incubate the embryos overnight.Embryos are contacted with an Agrobacterium strain containing theappropriate vectors for Ti plasmid mediated transfer for 5-10 min, andthen plated onto co-cultivation media for 3 days (25° C. in the dark).After co-cultivation, explants are transferred to recovery period mediafor five days (at 25° C. in the dark). Explants are incubated inselection media for up to eight weeks, depending on the nature andcharacteristics of the particular selection utilized. After theselection period, the resulting callus is transferred to embryomaturation media, until the formation of mature somatic embryos isobserved. The resulting mature somatic embryos are then placed under lowlight, and the process of regeneration is initiated as known in the art.The resulting shoots are allowed to root on rooting media, and theresulting plants are transferred to nursery pots and propagated astransgenic plants.

Example 15 Transformation of Maize Cells with the Insecticidal Genes ofthe Invention

Maize ears are collected 8-12 days after pollination. Embryos areisolated from the ears, and those embryos 0.8-1.5 mm in size are usedfor transformation. Embryos are plated scutellum side-up on a suitableincubation media, such as DN62A5S media (3.98 g/L N6 Salts; 1 mL/L (of1000× Stock) N6 Vitamins; 800 mg/L L-Asparagine; 100 mg/L Myo-inositol;1.4 g/L L-Proline; 100 mg/L Casaminoacids; 50 g/L sucrose; 1 mL/L (of 1mg/mL Stock) 2,4-D), and incubated overnight at 25° C. in the dark.

The resulting explants are transferred to mesh squares (30-40 perplate), transferred onto osmotic media for 30-45 minutes, thentransferred to a beaming plate (see, for example, PCT Publication No.WO/0138514 and U.S. Pat. No. 5,240,842).

DNA constructs designed to express the genes of the invention in plantcells are accelerated into plant tissue using an aerosol beamaccelerator, using conditions essentially as described in PCTPublication No. WO/0138514. After beaming, embryos are incubated for 30min on osmotic media, then placed onto incubation media overnight at 25°C. in the dark. To avoid unduly damaging beamed explants, they areincubated for at least 24 hours prior to transfer to recovery media.Embryos are then spread onto recovery period media, for 5 days, 25° C.in the dark, then transferred to a selection media. Explants areincubated in selection media for up to eight weeks, depending on thenature and characteristics of the particular selection utilized. Afterthe selection period, the resulting callus is transferred to embryomaturation media, until the formation of mature somatic embryos isobserved. The resulting mature somatic embryos are then placed under lowlight, and the process of regeneration is initiated by methods known inthe art. The resulting shoots are allowed to root on rooting media, andthe resulting plants are transferred to nursery pots and propagated astransgenic plants.

Materials

DN62A5S Media Components per Liter Source Chu'S N6 Basal Salt 3.98 g/LPhytotechnology Labs Mixture (Prod. No. C 416) Chu's N6 Vitamin Solution1 mL/L Phytotechnology Labs (Prod. No. C 149) (of 1000x Stock)L-Asparagine 800 mg/L Phytotechnology Labs Myo-inositol 100 mg/L SigmaL-Proline 1.4 g/L Phytotechnology Labs Casaminoacids 100 mg/L FisherScientific Sucrose 50 g/L Phytotechnology Labs 2,4-D (Prod. No. D-7299)1 mL/L Sigma (of 1 mg/mL Stock)

Adjust the pH of the solution to pH to 5.8 with 1N KOH/1N KCl, addGelrite (Sigma) to 3 g/L, and autoclave. After cooling to 50° C., add 2ml/l of a 5 mg/ml stock solution of Silver Nitrate (PhytotechnologyLabs). This recipe yields about 20 plates.

All publications and patent applications mentioned in the specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

That which is claimed:
 1. An isolated or recombinant nucleic acidmolecule comprising a nucleotide sequence encoding an insecticidalpolypeptide having at least 95% sequence identity SEQ ID NO:6.
 2. Thenucleic acid molecule of claim 1, wherein said nucleotide sequence isselected from the group consisting of SEQ ID NO:15 and
 16. 3. Anexpression cassette comprising the nucleic acid molecule of claim
 1. 4.The expression cassette of claim 3, further comprising a nucleic acidmolecule encoding a heterologous polypeptide.
 5. A host cell thatcontains the recombinant nucleic acid molecule of claim
 1. 6. Anisolated polypeptide having at least 95% sequence identity SEQ ID NO:6,wherein said polypeptide has insecticidal activity.
 7. The polypeptideof claim 6 further comprising heterologous amino acid sequences.
 8. Anantibody that selectively binds to the polypeptide of claim
 6. 9. Acomposition comprising the polypeptide of claim
 6. 10. The compositionof claim 9, wherein said composition is selected from the groupconsisting of a powder, dust, pellet, granule, spray, emulsion, colloid,and solution.
 11. The composition of claim 9, wherein said compositionis prepared by desiccation, lyophilization, homogenization, extraction,filtration, centrifugation, sedimentation, or concentration of a cultureof Bacillus thuringiensis cells.
 12. The composition of claim 9,comprising from about 1% to about 99% by weight of said polypeptide. 13.A plant having stably incorporated into its genome a DNA constructcomprising a nucleotide sequence encoding an insecticidal polypeptidehaving at least 95% sequence identity SEQ ID NO:6, wherein saidnucleotide sequence is operably linked to a promoter that drivesexpression of a coding sequence in a plant cell.
 14. The plant of claim13, wherein said plant is a plant cell.
 15. A transgenic seed of theplant of claim
 13. 16. The plant of claim 13, wherein said plant isselected from the group consisting of maize, sorghum, wheat, cabbage,sunflower, tomato, crucifers, peppers, potato, cotton, rice, soybean,sugarbeet, sugarcane, tobacco, barley, and oilseed rape.
 17. A methodfor protecting a plant from an insect pest, comprising introducing intosaid plant or cell thereof at least one expression vector comprising thenucleic acid molecule of claim
 1. 18. The nucleic acid molecule of claim1, wherein said nucleotide sequence is operably linked to a promotercapable of directing expression of said nucleotide sequence in a plantcell.